Methods of treating cea-positive cancers using pd-1 axis binding antagonists and anti-cea/anti-cd3 bispecific antibodies

ABSTRACT

The invention provides compositions and methods for treating CEA-positive cancers. The method comprising administering a PD-1 axis binding antagonist and a bispecific antibody that targets CEA and CD3.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of U.S. patent application Ser. No.15/399,122, filed on Jan. 5, 2017, which claims priority to EuropeanPatent Application No. 16150506.0, filed Jan. 8, 2016, the disclosuresof which are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The present application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Feb. 10, 2020, isnamed 51177-019002_Sequence_Listing_2.10.20_ST25 and is 56,016 bytes insize.

FIELD OF THE INVENTION

This invention relates to methods of treating CEA-positive cancers byadministering a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody.

BACKGROUND OF THE INVENTION

Cancer is one of the leading causes of death worldwide. Despite advancesin treatment options, prognosis of patients with advanced cancer remainspoor. Consequently, there is a persisting and urgent medical need foroptimal therapies to increase survival of cancer patients withoutcausing unacceptable toxicity.

Recent results from clinical trials have shown that immune therapies,particularly immune checkpoint inhibitors, can extend the overallsurvival of cancer patients and lead to durable responses. Despite thesepromising results, current immune-based therapies are only effective ina proportion of patients and combination strategies are needed toimprove therapeutic benefit.

Programmed death-ligand 1 (PD-L1) is found on the surface of immune andtumor cells and its expression is induced by interferon gamma (IFNγ). Itprevents the immune system from destroying cancer cells by interactingwith the inhibitory programmed death-1 (PD-1) and B7.1 receptors onactivated T cells, which results in a T-cell inhibitory signal.

As a result, therapeutic targeting of PD-1 and other molecules whichsignal through interactions with PD-1, such as programmed death-ligand 1(PD-L1) and programmed death ligand 2 (PD-L2) are an area of intenseinterest. PD-L1 is overexpressed in many cancers and is often associatedwith poor prognosis (Okazaki T et al., Intern. Immun. 2007 19(7):813)(Thompson R H et al., Cancer Res 2006, 66(7):3381). Interestingly, themajority of tumor infiltrating T lymphocytes predominantly express PD-1,in contrast to T lymphocytes in normal tissues and peripheral blood Tlymphocytes indicating that up-regulation of PD-1 on tumor-reactive Tcells can contribute to impaired antitumor immune responses (Blood 2009114(8):1537). This may be due to exploitation of PD-L1 signalingmediated by PD-L1 expressing tumor cells interacting with PD-1expressing T cells to result in attenuation of T cell activation andevasion of immune surveillance (Sharpe et al., Nat Rev 2002) (Keir M Eet al., 2008 Annu. Rev. Immunol. 26:677). Therefore, inhibition of thePD-L1/PD-1 interaction may enhance CD8+ T cell-mediated killing oftumors.

The inhibition of PD-L1 signaling has been proposed as a means toenhance T cell immunity for the treatment of cancer (e.g., tumorimmunity) and infection, including both acute and chronic (e.g.,persistent) infection. An optimal therapeutic treatment may combineblockade of PD-1 receptor/ligand interaction with an agent thatactivates T cells, particularly CD8+ T cells.

Dual blockade of PD-1 and PD-L1 has been reported to improve T cellkilling of tumor directed by CEA BiTE in an in vitro model (Osada etal., Cancer Immunol Immunother (2015) 64(6):677-88).

There remains a need for optimal therapies for treating, stabilizing,preventing, and/or delaying development of various cancers in patients.

All references cited herein, including patent applications, patentpublications, and UniProtKB/Swiss-Prot Accession numbers are hereinincorporated by reference in their entirety, as if each individualreference were specifically and individually indicated to beincorporated by reference.

SUMMARY OF THE INVENTION

In one aspect, provided herein is a method for treating or delayingprogression of cancer in an individual comprising administering to theindividual an effective amount of a human PD-1 axis binding antagonistand an anti-CEA/anti-CD3 bispecific antibody.

In another aspect, provided herein is a method of enhancing immunefunction in an individual having cancer comprising administering aneffective amount of a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody.

In another aspect, provided herein is use of a human PD-1 axis bindingantagonist in the manufacture of a medicament for treating or delayingprogression of cancer in an individual, wherein the medicament comprisesthe human PD-1 axis binding antagonist and an optional pharmaceuticallyacceptable carrier, and wherein the treatment comprises administrationof the medicament in combination with a composition comprising ananti-CEA/anti-CD3 bispecific antibody and an optional pharmaceuticallyacceptable carrier.

In another aspect, provided herein is use of an anti-CEA/anti-CD3bispecific antibody in the manufacture of a medicament for treating ordelaying progression of cancer in an individual, wherein the medicamentcomprises the anti-CEA/anti-CD3 bispecific antibody and an optionalpharmaceutically acceptable carrier, and wherein the treatment comprisesadministration of the medicament in combination with a compositioncomprising a human PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier.

In another aspect, provided herein is a composition comprising a humanPD-1 axis binding antagonist and an optional pharmaceutically acceptablecarrier for use in treating or delaying progression of cancer in anindividual, wherein the treatment comprises administration of saidcomposition in combination with a second composition, wherein the secondcomposition comprises an anti-CEA/anti-CD3 bispecific antibody and anoptional pharmaceutically acceptable carrier.

In another aspect, provided herein is a composition comprising ananti-CEA/anti-CD3 bispecific antibody and an optional pharmaceuticallyacceptable carrier for use in treating or delaying progression of cancerin an individual, wherein the treatment comprises administration of saidcomposition in combination with a second composition, wherein the secondcomposition comprises a human PD-1 axis binding antagonist and anoptional pharmaceutically acceptable carrier.

In another aspect, provided herein is a kit comprising a medicamentcomprising a PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising an anti-CEA/anti-CD3 bispecific antibody and anoptional pharmaceutically acceptable carrier for treating or delayingprogression of cancer in an individual.

In another aspect, provided herein is a kit comprising a firstmedicament comprising a PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier, and a second medicament comprisingan anti-CEA/anti-CD3 bispecific antibody and an optionalpharmaceutically acceptable carrier. In some embodiments, the kitfurther comprises a package insert comprising instructions foradministration of the first medicament and the second medicament fortreating or delaying progression of cancer in an individual.

In another aspect, provided herein is a kit comprising a medicamentcomprising an anti-CEA/anti-CD3 bispecific antibody and an optionalpharmaceutically acceptable carrier, and a package insert comprisinginstructions for administration of the medicament in combination with acomposition comprising a PD-1 axis binding antagonist and an optionalpharmaceutically acceptable carrier for treating or delaying progressionof cancer in an individual.

In some embodiments of the methods, uses, compositions, and kitsdescribed above and herein, the PD-1 axis binding antagonist is selectedfrom the group consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist. In some embodiments, the PD-1axis binding antagonist is an antibody. In some embodiments, theantibody is a humanized antibody, a chimeric antibody or a humanantibody. In some embodiments, the antibody is an antigen bindingfragment. In some embodiments, the antigen-binding fragment is selectedfrom the group consisting of Fab, Fab′, F(ab′)₂, and Fv.

In some embodiments, the PD-1 axis binding antagonist is a PD-1 bindingantagonist. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to its ligand binding partners. In some embodiments,the PD-1 binding antagonist inhibits the binding of PD-1 to PD-L1. Insome embodiments, the PD-1 binding antagonist inhibits the binding ofPD-1 to PD-L2. In some embodiments, the PD-1 binding antagonist inhibitsthe binding of PD-1 to both PD-L1 and PD-L2. In some embodiments, thePD-1 binding antagonist is an antibody. In some embodiments, the PD-1binding antagonist is selected from the group consisting of MDX 1106(nivolumab), MK-3475 (pembrolizumab), CT-011 (pidilizumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108.

In some embodiments, the PD-1 axis binding antagonist is a PD-L1 bindingantagonist. In some embodiments, the PD-L1 binding antagonist inhibitsthe binding of PD-L1 to PD-1. In some embodiments, the PD-L1 bindingantagonist inhibits the binding of PD-L1 to B7-1. In some embodiments,the PD-L1 binding antagonist inhibits the binding of PD-L1 to both PD-1and B7-1. In some embodiments, the PD-L1 binding antagonist is ananti-PD-L1 antibody. In some embodiments, the PD-L1 binding antagonistis selected from the group consisting of: MPDL3280A (atezolizumab),YW243.55.S70, MDX-1105, MEDI4736 (durvalumab), and MSB0010718C(avelumab). In some embodiments, the anti-PD-L1 antibody is MPDL3280A(atezolizumab). In some embodiments, MPDL3280A is administered at a doseof about 800 mg to about 1500 mg every three weeks (e.g., about 1000 mgto about 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mgevery three weeks). In some embodiments, MPDL3280A is administered at adose of about 1200 mg every three weeks. In some embodiments, theanti-PD-L1 antibody comprises a heavy chain comprising HVR-H1 sequenceof SEQ ID NO:19, HVR-H2 sequence of SEQ ID NO:20, and HVR-H3 sequence ofSEQ ID NO:21; and/or a light chain comprising HVR-L1 sequence of SEQ IDNO:22, HVR-L2 sequence of SEQ ID NO:23, and HVR-L3 sequence of SEQ IDNO:24. In some embodiments, the anti-PD-L1 antibody comprises a heavychain variable region comprising the amino acid sequence of SEQ ID NO:25or 26 and/or a light chain variable region comprising the amino acidsequence of SEQ ID NO:4. In some embodiments, the anti-PD-L1 antibodycomprises a heavy chain variable region comprising the amino acidsequence of SEQ ID NO:25 and a light chain variable region comprisingthe amino acid sequence of SEQ ID NO:4. In some embodiments, theanti-PD-L1 antibody comprises the three heavy chain HVR sequences ofantibody YW243.55.570 and/or the three light chain HVR sequences ofantibody YW24355.570 described in WO 2010/077634 and U.S. Pat. No.8,217,149, which are incorporated herein by reference. In someembodiments, the anti-PD-L1 antibody comprises the heavy chain variableregion sequence of antibody YW243.55.570 and/or the light chain variableregion sequence of antibody YW24355.570.

In some embodiments, the PD-1 axis binding antagonist is a PD-L2 bindingantagonist. In some embodiments, the PD-L2 binding antagonist is anantibody. In some embodiments, the PD-L2 binding antagonist is animmunoadhesin.

In some embodiments, the PD-1 axis binding antagonist is an antibody(e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody, or an anti-PD-L2antibody) and comprises an aglycosylation site mutation. In someembodiments, the aglycosylation site mutation is a substitutionmutation. In some embodiments, the substitution mutation is at aminoacid residue N297, L234, L235, and/or D265 (EU numbering). In someembodiments, the substitution mutation is selected from the groupconsisting of N297G, N297A, L234A, L235A, and D265A. In someembodiments, the substitution mutation is a D265A mutation and an N297Gmutation. In some embodiments, the aglycosylation site mutation reduceseffector function of the antibody. In some embodiments, the PD-1 axisbinding antagonist (e.g., an anti-PD-1 antibody, an anti-PD-L1 antibody,or an anti-PD-L2 antibody) is a human IgG₁ having Asn to Alasubstitution at position 297 according to EU numbering.

In some embodiments of the methods, uses, compositions, and kitsdescribed above and herein, the anti-CEA/anti-CD3 bispecific antibodycomprises a first antigen binding domain that binds to CD3, and a secondantigen binding domain that binds to CEA. In some embodiments, theanti-CEA/anti-CD3 bispecific antibody further comprises a third antigenbinding domain that binds to CEA. In some embodiments, the first antigenbinding domain binds to a human CD3 polypeptide. In some embodiments,the CD3 polypeptide is a human CDR polypeptide or a human CD3γpolypeptide. In some embodiments, the CD3 polypeptide is a human CD3εpolypeptide. In some embodiments, the first antigen binding domain bindsto a human CDR polypeptide or a human CD3 γ polypeptide in native T-cellreceptor (TCR) complex in association with other TCR subunits. In someembodiments, the first antigen binding domain comprises a heavy chainvariable region (V_(H)CD3) and a light chain variable region (V_(L)CD3),and the second (and third, if present) antigen binding domain comprisesa heavy chain variable region (V_(H)CEA) and a light chain variableregion (V_(L)CEA). In some embodiments, the first antigen binding domaincomprises a heavy chain variable region (V_(H)CD3) comprising HVR-H1sequence of SEQ ID NO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3sequence of SEQ ID NO:46; and/or a light chain variable region(V_(L)CD3) comprising HVR-L1 sequence of SEQ ID NO:47, HVR-L2 sequenceof SEQ ID NO:48, and HVR-L3 sequence of SEQ ID NO:49. In someembodiments, the first antigen binding domain comprises a heavy chainvariable region (V_(H)CD3) comprising the amino acid sequence of SEQ IDNO:50 and/or a light chain variable region (V_(L)CD3) comprising theamino acid sequence of SEQ ID NO:51. In some embodiments, the second(and third, if present) antigen binding domain comprises a heavy chainvariable region (V_(H)CEA) comprising HVR-H1 sequence of SEQ ID NO:38,HVR-H2 sequence of SEQ ID NO:39, and HVR-H3 sequence of SEQ ID NO:40;and/or a light chain variable region (V_(L)CEA) comprising HVR-L1sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ ID NO:42, and HVR-L3sequence of SEQ ID NO:43. In some embodiment, the second (and third, ifpresent) antigen binding domain comprises a heavy chain variable region(V_(H)CEA) comprising the amino acid sequence of SEQ ID NO:34 and/or alight chain variable region (V_(L)CEA) comprising the amino acidsequence of SEQ ID NO:35.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprisesa first antigen binding domain that binds to CD3 and a second andoptionally a third antigen binding domain that binds to CEA, wherein theantigen binding domains are Fab molecules. In particular suchembodiments, the bispecific antibody comprises a first antigen bindingdomain that binds to CD3 and a second and optionally a third antigenbinding domain that binds to CEA, wherein the first antigen bindingdomain is a crossover Fab molecule wherein the variable domains or theconstant domains of the Fab heavy and light chain are exchanged (i.e.replaced by each other) and the second (and third, if present) antigenbinding domain is a conventional Fab molecule. In one embodiment, thefirst antigen binding domain is a crossover Fab molecule wherein theconstant domains of the Fab heavy and light chain are exchanged. In oneembodiment, the anti-CEA/anti-CD3 bispecific antibody comprises not morethan one antigen binding domain that binds to CD3. In one embodiment,the first and the second antigen binding domain are fused to each other,optionally through a peptide linker. In some embodiments, theanti-CEA/anti-CD3 bispecific antibody comprises a first antigen bindingdomain that binds to CD3 and a second and optionally a third antigenbinding domain that binds to CEA, wherein the antigen binding domainsare Fab molecules and (i) the second antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain, or (ii) the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding domain.In a particular embodiment, the second antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding domain.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody furthercomprises an Fc domain composed of a first and a second subunit capableof stable association. In some embodiments, the anti-CEA/anti-CD3bispecific antibody comprises a first antigen binding domain that bindsto CD3 and a second and optionally a third antigen binding domain thatbinds to CEA, wherein the antigen binding domains are Fab molecules and(i) the second antigen binding domain is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the firstantigen binding domain, and the first antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain, or (ii) the first antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding domain,and the second antigen binding domain is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or second subunit of theFc domain. In a particular embodiment, the second antigen binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding domain, and the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or second subunit of the Fc domain.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprisesa first antigen binding domain that binds to CD3 and a second and athird antigen binding domain that binds to CEA, wherein the antigenbinding domains are Fab molecules and (i) the second antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding domain,the first antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the first subunit of the Fc domain, andthe third antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the second subunit of the Fc domain, or(ii) the first antigen binding domain is fused at the C-terminus of theFab heavy chain to the N-terminus of the Fab heavy chain of the secondantigen binding domain, the second antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and the third antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. In a particular embodiment, the second antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding domain,the first antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the first subunit of the Fc domain, andthe third antigen binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the second subunit of the Fc domain.

In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises

(i) a first antigen binding domain that binds to CD3, comprising a heavychain variable region (V_(H)CD3) comprising HVR-H1 sequence of SEQ IDNO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ IDNO:46; and a light chain variable region (V_(L)CD3) comprising HVR-L1sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3sequence of SEQ ID NO:49, wherein the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding domain that bind to CEA,comprising a heavy chain variable region (V_(H)CEA) comprising HVR-H1sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3sequence of SEQ ID NO:40; and a light chain variable region (V_(L)CEA)comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ IDNO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and thirdantigen binding moiety are each a Fab molecule, particularly aconventional Fab molecule;(iii) an Fc domain composed of a first and a second subunit capable ofstable association, wherein the second antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding domain, and the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.

In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3bispecific antibody is an IgG, specifically an IgG₁ or IgG₄, Fc domain.In some embodiments, the Fc domain is a human Fc domain. In oneembodiment, the Fc domain is a human IgG₁ Fc domain.

In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3bispecific antibody comprises a modification promoting the associationof the first and the second subunit of the Fc domain. In some suchembodiments, in the CH3 domain of the first subunit of the Fc domain anamino acid residue is replaced with an amino acid residue having alarger side chain volume, thereby generating a protuberance within theCH3 domain of the first subunit which is positionable in a cavity withinthe CH3 domain of the second subunit, and in the CH3 domain of thesecond subunit of the Fc domain an amino acid residue is replaced withan amino acid residue having a smaller side chain volume, therebygenerating a cavity within the CH3 domain of the second subunit withinwhich the protuberance within the CH3 domain of the first subunit ispositionable.

In some embodiments, the Fc domain comprised in the anti-CEA/anti-CD3bispecific antibody exhibits reduced binding affinity to an Fc receptorand/or reduced effector function, as compared to a native IgG₁ Fcdomain. In some embodiments, the Fc receptor is an Fcγ receptor and/orthe effector function is antibody-dependent cell-mediated cytotoxicity(ADCC). In some such embodiments, the Fc domain comprises one or moreamino acid substitution that reduces binding to an Fc receptor and/oreffector function. In one embodiment, said one or more amino acidsubstitution is at one or more position selected from the group of L234,L235, and P329 (EU numbering). In one embodiment, each subunit of the Fcdomain comprises three amino acid substitutions that reduce binding toan activating Fc receptor and/or effector function wherein said aminoacid substitutions are L234A, L235A and P329G (EU numbering).

In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In oneembodiment, the bispecific antibody comprises a polypeptide comprisingthe sequence of SEQ ID NO: 52, a polypeptide comprising the sequence ofSEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54,and a polypeptide comprising the sequence of SEQ ID NO: 55. (CEA TCB)

In particular embodiments, the anti-CEA/anti-CD3 bispecific antibody isCEA TCB. In some embodiments, CEA TCB is administered at a dose of about5 mg to about 400 mg every week (e.g., about 10 mg to about 60 mg everyweek, e.g., about 10 mg to about 40 mg every week, e.g. about 80 mg toabout 200 mg every week, e.g. about 80 mg to about 400 mg every week, ore.g. about 160 mg to 400 mg every week). In some embodiments, CEA TCB isadministered at a dose of about 5 mg, about 10 mg, about 20 mg, about 40mg, about 80 mg about 160 mg, particular at least about 80 mg, everyweek.

In some embodiments of the methods, uses, compositions and kitsdescribed above and herein, the cancer is a CEA-positive cancer. In someembodiments, the cancer is colon cancer, lung cancer, ovarian cancer,gastric cancer, bladder cancer, pancreatic cancer, endometrial cancer,breast cancer, kidney cancer, esophageal cancer, prostate cancer, orother cancers described herein. In some embodiments, the individual hascancer or has been diagnosed with cancer. In some embodiments, theindividual has locally advanced or metastatic cancer or has beendiagnosed with locally advanced or metastatic cancer. In someembodiments, the cancer cells in the individual express PD-L1. In someembodiments, the expression of PD-L1 may be determined by animmunohistochemistry (IHC) assay.

In some embodiments of the methods, uses, compositions, and kitsdescribed above and herein, the treatment or administration of the humanPD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecificantibody may result in a response in the individual. In someembodiments, the response is a complete response. In some embodiments,the response is a sustained response after cessation of the treatment.In some embodiments, the response is a complete response that issustained after cessation of the treatment. In other embodiments, theresponse is a partial response. In some embodiments, the response is apartial response that is sustained after cessation of the treatment.

In some embodiments of the methods, uses, compositions, and kitsdescribed above and herein, the anti-CEA/anti-CD3 bispecific antibody isadministered before the PD-1 axis binding antagonist, simultaneous withthe PD-1 axis binding antagonist, or after the PD-1 axis bindingantagonist. In some embodiments, the anti-CEA/anti-CD3 bispecificantibody is administered after the PD-1 axis binding antagonist,particularly at least one hour after the PD-1 axis binding antagonistwhen they are both administered on the same day. In some embodiments,the treatment or administration of the human PD-1 axis bindingantagonist and the anti-CEA/anti-CD3 bispecific antibody comprises adosing regimen comprising treatment cycles, wherein the individual isadministered, on day 1 of each cycle, a human PD-1 axis bindingantagonist at a dose of about 1200 mg, and on days 1, 8, and 15 of eachcycle, an anti-CEA/anti-CD3 antibody at a dose of about 5 mg, about 10mg, about 20 mg, about 40 mg, about 80 mg about 160 mg, particular atleast about 80 mg, each cycle being repeated every 21 days. In someembodiments, the cycle is repeated until there is loss of clinicalbenefit and/or unacceptable toxicity. In some embodiments, cycles arerepeated in which only the PD-1 axis binding antagonist or only theanti-CEA/anti-CD3 antibody is administered.

In some embodiments, the PD-1 axis binding antagonist and theanti-CEA/anti-CD3 bispecific antibody are in the same composition. Insome embodiments, the PD-1 axis binding antagonist and theanti-CEA/anti-CD3 bispecific antibody are in separate compositions.

In some embodiments of the methods, uses, compositions, and kitsdescribed above and herein, the PD-1 axis binding antagonist and/or theanti-CEA/anti-CD3 bispecific antibody is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. In some embodiments,the PD-1 axis binding antagonist and/or the anti-CEA/anti-CD3 bispecificantibody is administered intravenously. In some embodiments of themethods, uses, compositions, and kits described above and herein, thetreatment further comprises administering a chemotherapeutic agent fortreating or delaying progression of cancer in an individual. In someembodiments, the individual has been treated with a chemotherapeuticagent before the combination treatment with the PD-1 axis bindingantagonist and the anti-CEA/anti-CD3 bispecific antibody. In someembodiments, the individual treated with the combination of the PD-1axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibodyis refractory to a chemotherapeutic agent treatment. In someembodiments, the individual treated with the combination of the PD-1axis binding antagonist and/or the anti-CEA/anti-CD3 bispecific antibodyis intolerant to a chemotherapeutic agent treatment. Some embodiments ofthe methods, uses, compositions, and kits described throughout theapplication, further comprise administering a chemotherapeutic agent fortreating or delaying progression of cancer.

In some embodiments of the methods, uses, compositions and kitsdescribed above and herein, CD8 T cells in the individual have enhancedpriming, activation, proliferation and/or cytolytic activity relative toprior to the administration of the combination. In some embodiments, thenumber of CD8 T cells is elevated relative to prior to administration ofthe PD-1 axis binding antagonist and the anti-CEA/anti-CD3 antibody. Insome embodiments, the CD8 T cell is an antigen-specific CD8 T cell. Insome embodiments, Treg function is suppressed relative to prior to theadministration of the PD-1 axis binding antagonist and theanti-CEA/anti-CD3 antibody. In some embodiments, T cell exhaustion isdecreased relative to prior to the administration of the PD-1 axisbinding antagonist and the anti-CEA/anti-CD3 antibody.

It is to be understood that one, some, or all of the properties of thevarious embodiments described herein may be combined to form otherembodiments of the present invention. These and other aspects of theinvention will become apparent to one of skill in the art. These andother embodiments of the invention are further described by the detaileddescription that follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. CEA TCB-mediated lysis of MKN45 cells. Representativegraphs of CEA TCB-mediated lysis of MKN-45 tumor cells assessed (A) 24hours and (B) 48 hours after incubation of tumor cells with human PBMCs(E:T 10:1). The EC50 values of tumor cell killing are: 615 pM (24 h),362 pM (48 h). Target cell killing was assessed by quantification of LDHreleased into cell supernatants.

FIGS. 2A-2C. CEA TCB-mediated up-regulation of PD-1 on T cells, and ofPD-L1 on surviving tumor cells after tumor cell lysis. Representativegraphs of CEA TCB-mediated up-regulation of PD-1 receptor expression onCD8+ T cells (A), CD4+ T cells (B), and of PD-L1 ligand expression ontumor cells that resisted killing (C) analyzed 48 h post incubation ofMKN-45 tumor cells with human PBMCs (E:T 10:1). PD-1 and PD-L1expression was analyzed by flow cytometry.

FIGS. 3A and 3B. Tumor growth after treatment with CEA TCB (co-grafting,E:T 5:1). Average tumor burden and standard error of mean (SEM, n=12);tumor burden was measured by bioluminescence (total flux). CEA TCB wasadministered IV at doses of 0.5 and 2.5 mg/kg, starting one day (earlytreatment, panel A) or seven days (late treatment, panel B) aftertumor/PBMCs co-grafting and SC injection. Control groups receivedphosphate-buffer saline (PBS, vehicle), MCSP TCB, or DP47 TCB (asuntargeted controls, respectively). In all studies, TCBs wereadministered twice a week (2q7d). Arrows indicate the day of start oftherapy. The curves were compared using non-linear regression analysis.(A) Start of therapy one day after co-grafting with human PBMCs. Curvecomparison: vehicle vs. untargeted TCB p-value=0.65, vehicle vs. CEA TCB(0.5 mg/kg) p-value<0.0001, vehicle vs. CEA TCB (2.5 mg/kg)p-value<0.0001, CEA TCB (2.5 mg/kg) vs. CEA TCB (0.5 mg/kg)p-value=0.91. (B) Start of therapy seven days after co-grafting withhuman PBMCs. Curve comparison: vehicle vs. untargeted TCB p-value=0.65,vehicle vs. CEA TCB (0.5 mg/kg) p-value<0.0001, vehicle vs. CEA TCB (2.5mg/kg) p-value<0.0001, CEA TCB (2.5 mg/kg) vs. CEA TCB (0.5 mg/kg)p-value=0.0008.

FIGS. 4A-4F. Histological analysis of tumors after treatment with CEATCB (co-grafting, E:T 5:1). Hematoxylin and eosin (H&E, panels A and D)and anti-PD-L1 (panels B, C, E and F) staining of vehicle (panels A-C)and CEA TCB (panels D-F)-treated tumors (2.5 mg/kg, therapy given on Day7) collected at termination in the co-grafting experiment ofLS174T-fluc2 cells with hPBMCs (E:T 5:1) showing strong induction ofintra-tumor PD-L1 expression upon CEA TCB-treatment. Panel B and Ecorrespond to the marked area of panel A and D, respectively. Panel Cand F correspond to the marked area of panel B and E, respectively.

FIG. 5. Tumor weight after treatment with CEA TCB. LS174T-fluc2 cellsinjected SC and grown until a volume of 100 mm³ was reached in NOG mice,followed by IP transfer of human PBMCs (10×10⁶ cells). Three days afterPBMCs transfer, mice received bi-weekly IV injections of CEA TCB (2.5mg/kg), PBS (vehicle), or MCSP TCB (untargeted control, 2.5 mg/kg).Tumor mass was determined at study termination by weighing the explantedtumors. Graphs show average tumor weight and standard error of mean(SEM, 3≤n≤6 mice). ** p<0.005 using unpaired, two-tailed t-test.

FIG. 6. Flow cytometry analysis of tumor-infiltrating leukocytes aftertreatment with CEA TCB. Flow cytometry analysis of tumor tissuescollected at termination in the human colon carcinoma xenograft modelwith IP transfer of human effector cells. The bar plot shows the numberof intra-tumor human CD45+/CD3+ T-cells as assessed by flow cytometry inall treated groups. * p<0.05 using unpaired, two-tailed t-test.

FIGS. 7A-7L. Immuno PD analysis after treatment with CEA TCB (IPtransfer of human PBMCs). Flow cytometry analysis of blood (A-F) andtumor tissues (G-L) collected at Day 18 (corresponding to three daysafter the third CEA TCB administration) after tumor cell inoculation inthe human colon carcinoma xenograft model (IP transfer of human effectorcells). Dot plots show the expression of CD69 (B, E, H, K) and PD-1 (C,F, I, L) on CD3 T-cells in both compartments. Representative mice foreach group are shown. A-C, G-I: vehicle. D-F, J-L: CEA TCB

FIG. 8. CEA TCB-induced tumor growth inhibition in fully humanized mice.Tumor growth inhibition of MKN45 in humanized mice. Tumor volumes atstudy termination (at day 32 after tumor cell injection) are shown. Eachpoint represents an individual animal. CEA TCB was injected IV (2.5mg/kg twice weekly), and tumor growth was measured by caliper.* p<0.05(unpaired t-test).

FIGS. 9A-9D. Increase of intra-tumoral human T-cell frequencies andT-cell activation by CEA TCB. Flow cytometric analysis of tumor samplescollected at study termination (Day 32). Cells were stained for humanantigens (huCD45, huCD3, huPD-1, huCD69, huKi-67, and huGZB). (A, B)Total human T-cell frequencies (A) and the ratio of CD8+ to CD4+ T-cells(B) in the tumor tissue. (C, D) The expression of activation markers intumor is shown as percent of human CD8+(C) or CD4+(D) T-cells. Eachpoint represents an individual animal. * p<0.05, ** p<0.01, *** p<0.001,**** p<0.0001 (unpaired t-test).

FIGS. 10A-10F. Histological analyses of tumor tissues at studytermination. Hematoxylin and eosin (H&E, panel A and D) and anti-PD-L1(panel B, C, E and F) staining (Mab SP142) of vehicle (panel A-C) andCEA TCB (panel D-F)-treated tumors exhibiting strong induction ofintra-tumoral PD-L1 expression upon CEA TCB treatment. All sections werecounterstained with hematoxylin (blue nuclei). Panel B and E correspondto the marked area of panel A and D, respectively. Panel C and Fcorrespond to the marked area of panel B and E, respectively.

FIGS. 11A and 11B. In vivo anti-tumor activity upon combination of CEATCB with anti-human PD-L1 blocking antibody in MKN45 tumor model infully humanized mice. (A) Average tumor burden and standard error ofmean (SEM). Tumor burden was measured by digital caliper 3 times a week.Arrow indicates the day of start of therapy. At day 60: n=7 (vehicle),n=8 (CEA TCB); n=4 (a-PD-L1); n=5 (combination CEA TCB with anti-PD-L1).(B) Time-to-event statistical analysis. The event was defined asreaching 500 mm³ tumor volume. The pairwise log-rank test was used tocompare the following treatment groups, as it can take the differentdrop-outs into account: vehicle vs. CEA TCB: p=0.005; CEA TCB vs.anti-PD-L1: p=0.001; CEA TCB vs. combination of CEA TCB with anti-PD-L1:p=0.03.

FIG. 12. Anti-tumor activity of CEA TCB in the MKN45 model in fullyhumanized NOG mice. For first line treatment, mice were administeredeither with vehicle, as control, or with CEA TCB (2.5 mg/kg,administered twice a week for 4 weeks, 7 administrations in total). Atstudy day 34, mice in the CEA TCB-treated group were randomized into twosub-groups: one continued being administered with CEA TCB (2.5 mg/kgtwice a week) and the other was administered with CEA TCB (2.5 mg/kgtwice a week) in combination with anti-mouse PD-L1 antibody (10 mg/kg,administered once a week) for 5 weeks (11 administrations in total forCEA TCB and 6 administrations in total for anti-PD-L1). ***p<0.0002two-way ANOVA.

FIGS. 13A and 13B. Anti-tumor activity of CEA TCB in the MC38-huCEAmodel in fully immunocompetent huCEA Tg mice. (A) For first linetreatment, mice were administered either with vehicle, as control, withCEA TCB (2.5 mg/kg, administered twice a week for 4 weeks, 7administrations in total), with anti-mouse PD-L1 antibody (10 mg/kg,administered once a week for 4 weeks, 4 administrations in total), andwith a combination of CEA TCB and anti-mouse PD-L1, administeredconcomitantly with the respective dose and schedules used in the singletherapeutic groups. Treatment was administered for 4 weeks. (B)Sub-cutaneous tumors were surgically removed, and after 5 weeks micewere rechallenged with fresh MC38-huCEA tumors cells injectedsub-cutaneously on the opposite flank. As control, groups of naïve huCEATg mice, either left un-manipulated or submitted to a surgical cut onthe flank, were injected sub-cutaneously with MC38-huCEA on the oppositeflank.

DETAILED DESCRIPTION

The inventors of this application demonstrated that a CEA T cellbispecific antibody and anti-PD-L1 immune therapy act synergistically intheir anti-cancer properties and their combination could providemeaningful clinical benefit in patients with cancer. The data in theapplication show that the combination of a CEA T cell bispecificantibody (CEA TCB) with anti-PD-L1 immune therapy resulted in enhancedinhibition of tumor growth.

In one aspect, provided herein are methods, compositions and uses fortreating or delaying progression of cancer in an individual comprisingadministering an effective amount of a human PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody.

In another aspect, provided herein are methods, compositions and usesfor enhancing immune function in an individual having cancer comprisingadministering an effective amount of a human PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody.

I. Definitions

Before describing the invention in detail, it is to be understood thatthis invention is not limited to particular compositions or biologicalsystems, which can, of course, vary. It is also to be understood thatthe terminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

As used in this specification and the appended claims, the singularforms “a”, “an” and “the” include plural referents unless the contentclearly dictates otherwise. Thus, for example, reference to “a molecule”optionally includes a combination of two or more such molecules, and thelike.

As used herein, the terms “first”, “second”, “third” etc. with respectto antigen binding domains etc., are used for convenience ofdistinguishing when there is more than one of each type of domain. Useof these terms is not intended to confer a specific order or orientationunless explicitly so stated.

The term “about” as used herein refers to the usual error range for therespective value readily known to the skilled person in this technicalfield. Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and “consisting essentiallyof” aspects and embodiments.

The term “PD-1 axis binding antagonist” refers to a molecule thatinhibits the interaction of a PD-1 axis binding partner with either oneor more of its binding partner, so as to remove T-cell dysfunctionresulting from signaling on the PD-1 signaling axis—with a result beingto restore or enhance T-cell function (e.g., proliferation, cytokineproduction, target cell killing). As used herein, a PD-1 axis bindingantagonist includes a PD-1 binding antagonist, a PD-L1 bindingantagonist and a PD-L2 binding antagonist. A “human” PD-1 axis bindingantagonist refers to a PD-1 axis binding antagonist which has theabove-described effects on the human PD-1 signaling axis.

The term “PD-1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-1 with one or more of its bindingpartners, such as PD-L1, PD-L2. In some embodiments, the PD-1 bindingantagonist is a molecule that inhibits the binding of PD-1 to one ormore of its binding partners. In a specific aspect, the PD-1 bindingantagonist inhibits the binding of PD-1 to PD-L1 and/or PD-L2. Forexample, PD-1 binding antagonists include anti-PD-1 antibodies, antigenbinding fragments thereof, immunoadhesins, fusion proteins,oligopeptides and other molecules that decrease, block, inhibit,abrogate or interfere with signal transduction resulting from theinteraction of PD-1 with PD-L1 and/or PD-L2. In one embodiment, a PD-1binding antagonist reduces the negative co-stimulatory signal mediatedby or through cell surface proteins expressed on T lymphocytes mediatedsignaling through PD-1 so as render a dysfunctional T-cell lessdysfunctional (e.g., enhancing effector responses to antigenrecognition). In some embodiments, the PD-1 binding antagonist is ananti-PD-1 antibody. In a specific aspect, a PD-1 binding antagonist isMDX-1106 (nivolumab) described herein. In another specific aspect, aPD-1 binding antagonist is MK-3475 (pembrolizumab) described herein. Inanother specific aspect, a PD-1 binding antagonist is CT-011(pidilizumab) described herein. In another specific aspect, a PD-1binding antagonist is MEDI-0680 (AMP-514) described herein. In anotherspecific aspect, a PD-1 binding antagonist is PDR001 described herein.In another specific aspect, a PD-1 binding antagonist is REGN2810described herein. In another specific aspect, a PD-1 binding antagonistis BGB-108 described herein.

The term “PD-L1 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L1 with either one or more of itsbinding partners, such as PD-1, B7-1. In some embodiments, a PD-L1binding antagonist is a molecule that inhibits the binding of PD-L1 toits binding partners. In a specific aspect, the PD-L1 binding antagonistinhibits binding of PD-L1 to PD-1 and/or B7-1. In some embodiments, thePD-L1 binding antagonists include anti-PD-L1 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L1 withone or more of its binding partners, such as PD-1, B7-1. In oneembodiment, a PD-L1 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L1 so as torender a dysfunctional T-cell less dysfunctional (e.g., enhancingeffector responses to antigen recognition). In some embodiments, a PD-L1binding antagonist is an anti-PD-L1 antibody. In a specific aspect, ananti-PD-L1 antibody is YW243.55.S70 described herein. In anotherspecific aspect, an anti-PD-L1 antibody is MDX-1105 described herein. Instill another specific aspect, an anti-PD-L1 antibody is MPDL3280A(atezolizumab) described herein. In still another specific aspect, ananti-PD-L1 antibody is MDX-1105 described herein. In still anotherspecific aspect, an anti-PD-L1 antibody is YW243.55.S70 describedherein. In still another specific aspect, an anti-PD-L1 antibody isMEDI4736 (durvalumab) described herein. In still another specificaspect, an anti-PD-L1 antibody is MSB0010718C (avelumab) describedherein.

The term “PD-L2 binding antagonist” refers to a molecule that decreases,blocks, inhibits, abrogates or interferes with signal transductionresulting from the interaction of PD-L2 with either one or more of itsbinding partners, such as PD-1. In some embodiments, a PD-L2 bindingantagonist is a molecule that inhibits the binding of PD-L2 to one ormore of its binding partners. In a specific aspect, the PD-L2 bindingantagonist inhibits binding of PD-L2 to PD-1. In some embodiments, thePD-L2 antagonists include anti-PD-L2 antibodies, antigen bindingfragments thereof, immunoadhesins, fusion proteins, oligopeptides andother molecules that decrease, block, inhibit, abrogate or interferewith signal transduction resulting from the interaction of PD-L2 witheither one or more of its binding partners, such as PD-1. In oneembodiment, a PD-L2 binding antagonist reduces the negativeco-stimulatory signal mediated by or through cell surface proteinsexpressed on T lymphocytes mediated signaling through PD-L2 so as rendera dysfunctional T-cell less dysfunctional (e.g., enhancing effectorresponses to antigen recognition). In some embodiments, a PD-L2 bindingantagonist is an immunoadhesin.

The term “dysfunction” in the context of immune dysfunction, refers to astate of reduced immune responsiveness to antigenic stimulation. Theterm includes the common elements of both exhaustion and/or anergy inwhich antigen recognition may occur, but the ensuing immune response isineffective to control infection or tumor growth.

The term “dysfunctional”, as used herein, also includes refractory orunresponsive to antigen recognition, specifically, impaired capacity totranslate antigen recognition into down-stream T-cell effectorfunctions, such as proliferation, cytokine production (e.g., IL-2)and/or target cell killing.

The term “anergy” refers to the state of unresponsiveness to antigenstimulation resulting from incomplete or insufficient signals deliveredthrough the T-cell receptor (e.g. increase in intracellular Ca²⁺ in theabsence of ras-activation). T cell anergy can also result uponstimulation with antigen in the absence of co-stimulation, resulting inthe cell becoming refractory to subsequent activation by the antigeneven in the context of costimulation. The unresponsive state can oftenbe overriden by the presence of interleukin-2. Anergic T-cells do notundergo clonal expansion and/or acquire effector functions.

The term “exhaustion” refers to T cell exhaustion as a state of T celldysfunction that arises from sustained TCR signaling that occurs duringmany chronic infections and cancer. It is distinguished from anergy inthat it arises not through incomplete or deficient signaling, but fromsustained signaling. It is defined by poor effector function, sustainedexpression of inhibitory receptors and a transcriptional state distinctfrom that of functional effector or memory T cells. Exhaustion preventsoptimal control of infection and tumors. Exhaustion can result from bothextrinsic negative regulatory pathways (e.g., immunoregulatorycytokines) as well as cell intrinsic negative regulatory (costimulatory)pathways (PD-1, B7-H3, B7-H4, etc.).

“Enhancing T-cell function” means to induce, cause or stimulate a T-cellto have a sustained or amplified biological function, or renew orreactivate exhausted or inactive T-cells. Examples of enhancing T-cellfunction include: increased secretion of γ-interferon from CD8⁺ T-cells,increased proliferation, increased antigen responsiveness (e.g., viral,pathogen, or tumor clearance) relative to such levels before theintervention. In one embodiment, the level of enhancement is as least50%, alternatively 60%, 70%, 80%, 90%, 100%, 120%, 150%, 200%. Themanner of measuring this enhancement is known to one of ordinary skillin the art.

A “T cell dysfunctional disorder” is a disorder or condition of T-cellscharacterized by decreased responsiveness to antigenic stimulation. In aparticular embodiment, a T-cell dysfunctional disorder is a disorderthat is specifically associated with inappropriate increased signalingthrough PD-1. In another embodiment, a T-cell dysfunctional disorder isone in which T-cells are anergic or have decreased ability to secretecytokines, proliferate, or execute cytolytic activity. In a specificaspect, the decreased responsiveness results in ineffective control of apathogen or tumor expressing an immunogen. Examples of T celldysfunctional disorders characterized by T-cell dysfunction includeunresolved acute infection, chronic infection and tumor immunity.

“Tumor immunity” refers to the process in which tumors evade immunerecognition and clearance. Thus, as a therapeutic concept, tumorimmunity is “treated” when such evasion is attenuated, and the tumorsare recognized and attacked by the immune system. Examples of tumorrecognition include tumor binding, tumor shrinkage and tumor clearance.

“Immunogenicity” refers to the ability of a particular substance toprovoke an immune response. Tumors are immunogenic and enhancing tumorimmunogenicity aids in the clearance of the tumor cells by the immuneresponse. Examples of enhancing tumor immunogenicity include treatmentwith a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody.

“Sustained response” refers to the sustained effect on reducing tumorgrowth after cessation of a treatment. For example, the tumor size mayremain to be the same or smaller as compared to the size at thebeginning of the administration phase. In some embodiments, thesustained response has a duration at least the same as the treatmentduration, at least 1.5×, 2.0×, 2.5×, or 3.0× length of the treatmentduration.

The term “pharmaceutical formulation” refers to a preparation which isin such form as to permit the biological activity of the activeingredient to be effective, and which contains no additional componentswhich are unacceptably toxic to a subject to which the formulation wouldbe administered. Such formulations are sterile. “Pharmaceuticallyacceptable” excipients (vehicles, additives) are those which canreasonably be administered to a subject mammal to provide an effectivedose of the active ingredient employed.

As used herein, the term “treatment” refers to clinical interventiondesigned to alter the natural course of the individual or cell beingtreated during the course of clinical pathology. Desirable effects oftreatment include decreasing the rate of disease progression,ameliorating or palliating the disease state, and remission or improvedprognosis. For example, an individual is successfully “treated” if oneor more symptoms associated with cancer are mitigated or eliminated,including, but are not limited to, reducing the proliferation of (ordestroying) cancerous cells, decreasing symptoms resulting from thedisease, increasing the quality of life of those suffering from thedisease, decreasing the dose of other medications required to treat thedisease, and/or prolonging survival of individuals.

As used herein, “delaying progression of a disease” means to defer,hinder, slow, retard, stabilize, and/or postpone development of thedisease (such as cancer). This delay can be of varying lengths of time,depending on the history of the disease and/or individual being treated.As is evident to one skilled in the art, a sufficient or significantdelay can, in effect, encompass prevention, in that the individual doesnot develop the disease. For example, a late stage cancer, such asdevelopment of metastasis, may be delayed.

An “effective amount” is at least the minimum amount required to effecta measurable improvement or prevention of a particular disorder. Aneffective amount herein may vary according to factors such as thedisease state, age, sex, and weight of the patient, and the ability ofthe antibody to elicit a desired response in the individual. Aneffective amount is also one in which any toxic or detrimental effectsof the treatment are outweighed by the therapeutically beneficialeffects. For prophylactic use, beneficial or desired results includeresults such as eliminating or reducing the risk, lessening theseverity, or delaying the onset of the disease, including biochemical,histological and/or behavioral symptoms of the disease, itscomplications and intermediate pathological phenotypes presenting duringdevelopment of the disease. For therapeutic use, beneficial or desiredresults include clinical results such as decreasing one or more symptomsresulting from the disease, increasing the quality of life of thosesuffering from the disease, decreasing the dose of other medicationsrequired to treat the disease, enhancing effect of another medicationsuch as via targeting, delaying the progression of the disease, and/orprolonging survival. In the case of cancer or tumor, an effective amountof the drug may have the effect in reducing the number of cancer cells;reducing the tumor size; inhibiting (i.e., slow to some extent ordesirably stop) cancer cell infiltration into peripheral organs; inhibit(i.e., slow to some extent and desirably stop) tumor metastasis;inhibiting to some extent tumor growth; and/or relieving to some extentone or more of the symptoms associated with the disorder. An effectiveamount can be administered in one or more administrations. For purposesof this invention, an effective amount of drug, compound, orpharmaceutical composition is an amount sufficient to accomplishprophylactic or therapeutic treatment either directly or indirectly. Asis understood in the clinical context, an effective amount of a drug,compound, or pharmaceutical composition may or may not be achieved inconjunction with another drug, compound, or pharmaceutical composition.Thus, an “effective amount” may be considered in the context ofadministering one or more therapeutic agents, and a single agent may beconsidered to be given in an effective amount if, in conjunction withone or more other agents, a desirable result may be or is achieved.

As used herein, “in conjunction with” refers to administration of onetreatment modality in addition to another treatment modality. As such,“in conjunction with” refers to administration of one treatment modalitybefore, during, or after administration of the other treatment modalityto the individual.

A “disorder” is any condition that would benefit from treatmentincluding, but not limited to, chronic and acute disorders or diseasesincluding those pathological conditions which predispose the mammal tothe disorder in question.

The terms “cell proliferative disorder” and “proliferative disorder”refer to disorders that are associated with some degree of abnormal cellproliferation. In one embodiment, the cell proliferative disorder iscancer. In one embodiment, the cell proliferative disorder is a tumor.

“Tumor”, as used herein, refers to all neoplastic cell growth andproliferation, whether malignant or benign, and all pre-cancerous andcancerous cells and tissues. The terms “cancer”, “cancerous”, “cellproliferative disorder”, “proliferative disorder” and “tumor” are notmutually exclusive as referred to herein.

The terms “cancer” and “cancerous” refer to or describe thephysiological condition in mammals that is typically characterized byunregulated cell growth. Examples of cancer include but are not limitedto, carcinoma, lymphoma, blastoma, sarcoma, and leukemia or lymphoidmalignancies. More particular examples of such cancers include, but notlimited to, squamous cell cancer (e.g., epithelial squamous cellcancer), lung cancer including small-cell lung cancer, non-small celllung cancer, adenocarcinoma of the lung and squamous carcinoma of thelung, cancer of the peritoneum, hepatocellular cancer, gastric orstomach cancer including gastrointestinal cancer and gastrointestinalstromal cancer, pancreatic cancer, glioblastoma, cervical cancer,ovarian cancer, liver cancer, bladder cancer, cancer of the urinarytract, hepatoma, breast cancer, colon cancer, rectal cancer, colorectalcancer, endometrial or uterine carcinoma, salivary gland carcinoma,kidney or renal cancer, prostate cancer, vulval cancer, thyroid cancer,hepatic carcinoma, anal carcinoma, penile carcinoma, melanoma,superficial spreading melanoma, lentigo maligna melanoma, acrallentiginous melanomas, nodular melanomas, multiple myeloma and B-celllymphoma (including low grade/follicular non-Hodgkin's lymphoma (NHL);small lymphocytic (SL) NHL; intermediate grade/follicular NHL;intermediate grade diffuse NHL; high grade immunoblastic NHL; high gradelymphoblastic NHL; high grade small non-cleaved cell NHL; bulky diseaseNHL; mantle cell lymphoma; AIDS-related lymphoma; and Waldenstrom'sMacroglobulinemia); chronic lymphocytic leukemia (CLL); acutelymphoblastic leukemia (ALL); hairy cell leukemia; chronic myeloblasticleukemia; and post-transplant lymphoproliferative disorder (PTLD), aswell as abnormal vascular proliferation associated with phakomatoses,edema (such as that associated with brain tumors), Meigs' syndrome,brain, as well as head and neck cancer, and associated metastases. Incertain embodiments, cancers that are amenable to treatment by theantibodies of the invention include breast cancer, colorectal cancer,rectal cancer, non-small cell lung cancer, glioblastoma, non-Hodgkinslymphoma (NHL), renal cell cancer, prostate cancer, liver cancer,pancreatic cancer, soft-tissue sarcoma, kaposi's sarcoma, carcinoidcarcinoma, head and neck cancer, ovarian cancer, mesothelioma, andmultiple myeloma. In some embodiments, the cancer is selected from:small cell lung cancer, glioblastoma, neuroblastomas, melanoma, breastcarcinoma, gastric cancer, colorectal cancer (CRC), and hepatocellularcarcinoma. Yet, in some embodiments, the cancer is selected from:non-small cell lung cancer, colorectal cancer, glioblastoma and breastcarcinoma, including metastatic forms of those cancers. In someembodiments, the cancer is a CEA-positive cancer.

The term “cytotoxic agent” as used herein refers to any agent that isdetrimental to cells (e.g., causes cell death, inhibits proliferation,or otherwise hinders a cellular function). Cytotoxic agents include, butare not limited to, radioactive isotopes (e.g., At²¹¹, I¹³¹, I¹²⁵, Y⁹⁰,Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactive isotopes of Lu);chemotherapeutic agents; growth inhibitory agents; enzymes and fragmentsthereof such as nucleolytic enzymes; and toxins such as small moleculetoxins or enzymatically active toxins of bacterial, fungal, plant oranimal origin, including fragments and/or variants thereof. Exemplarycytotoxic agents can be selected from anti-microtubule agents, platinumcoordination complexes, alkylating agents, antibiotic agents,topoisomerase II inhibitors, antimetabolites, topoisomerase Iinhibitors, hormones and hormonal analogues, signal transduction pathwayinhibitors, non-receptor tyrosine kinase angiogenesis inhibitors,immunotherapeutic agents, proapoptotic agents, inhibitors of LDH-A,inhibitors of fatty acid biosynthesis, cell cycle signalling inhibitors,HDAC inhibitors, proteasome inhibitors, and inhibitors of cancermetabolism. In one embodiment the cytotoxic agent is a taxane. In oneembodiment the taxane is paclitaxel or docetaxel. In one embodiment thecytotoxic agent is a platinum agent. In one embodiment the cytotoxicagent is an antagonist of EGFR. In one embodiment the antagonist of EGFRis N-(3-ethynylphenyl)-6,7-bis(2-methoxyethoxy)quinazolin-4-amine (e.g.,erlotinib). In one embodiment the cytotoxic agent is a RAF inhibitor. Inone embodiment, the RAF inhibitor is a BRAF and/or CRAF inhibitor. Inone embodiment the RAF inhibitor is vemurafenib. In one embodiment thecytotoxic agent is a PI3K inhibitor.

“Chemotherapeutic agent” includes compounds useful in the treatment ofcancer. Examples of chemotherapeutic agents include erlotinib (TARCEVA®,Genentech/OSI Pharm.), bortezomib (VELCADE®, Millennium Pharm.),disulfiram, epigallocatechin gallate, salinosporamide A, carfilzomib,17-AAG (geldanamycin), radicicol, lactate dehydrogenase A (LDH-A),fulvestrant (FASLODEX®, Astra7eneca), sunitib (SUTENT®, Pfizer/Sugen),letrozole (FEMARA®, Novartis), imatinib mesylate (GLEEVEC®, Novartis),finasunate (VATALANIB®, Novartis), oxaliplatin (ELOXATIN®, Sanofi), 5-FU(5-fluorouracil), leucovorin, Rapamycin (Sirolimus, RAPAMUNE®, Wyeth),Lapatinib (TYKERB®, GSK572016, Glaxo Smith Kline), Lonafamib (SCH66336), sorafenib (NEXAVAR®, Bayer Labs), gefitinib (IRESSA®,Astra7eneca), AG1478, alkylating agents such as thiotepa and CYTOXAN®cyclosphosphamide; alkyl sulfonates such as busulfan, improsulfan andpiposulfan; aziridines such as benzodopa, carboquone, meturedopa, anduredopa; ethylenimines and methylamelamines including altretamine,triethylenemelamine, triethylenephosphoramide,triethylenethiophosphoramide and trimethylomelamine; acetogenins(especially bullatacin and bullatacinone); a camptothecin (includingtopotecan and irinotecan); bryostatin; callystatin; CC-1065 (includingits adozelesin, carzelesin and bizelesin synthetic analogs);cryptophycins (particularly cryptophycin 1 and cryptophycin 8);adrenocorticosteroids (including prednisone and prednisolone);cyproterone acetate; 5α-reductases including finasteride anddutasteride); vorinostat, romidepsin, panobinostat, valproic acid,mocetinostat dolastatin; aldesleukin, talc duocarmycin (including thesynthetic analogs, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; asarcodictyin; spongistatin; nitrogen mustards such as chlorambucil,chlomaphazine, chlorophosphamide, estramustine, ifosfamide,mechlorethamine, mechlorethamine oxide hydrochloride, melphalan,novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard;nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine,nimustine, and ranimnustine; antibiotics such as the enediyneantibiotics (e.g., calicheamicin, especially calicheamicin γ1I andcalicheamicin ω1I (Angew Chem. Intl. Ed. Engl. 1994 33:183-186);dynemicin, including dynemicin A; bisphosphonates, such as clodronate;an esperamicin; as well as neocarzinostatin chromophore and relatedchromoprotein enediyne antibiotic chromophores), aclacinomysins,actinomycin, authramycin, azaserine, bleomycins, cactinomycin,carabicin, caminomycin, carzinophilin, chromomycinis, dactinomycin,daunorubicin, detorubicin, 6-diazo-5-oxo-L-norleucine, ADRIAMYCIN®(doxorubicin), morpholino-doxorubicin, cyanomorpholino-doxorubicin,2-pyrrolino-doxorubicin and deoxydoxorubicin), epirubicin, esorubicin,idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolicacid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin,quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin,ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexateand 5-fluorouracil (5-FU); folic acid analogs such as denopterin,methotrexate, pteropterin, trimetrexate; purine analogs such asfludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidineanalogs such as ancitabine, azacitidine, 6-azauridine, carmofur,cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine;androgens such as calusterone, dromostanolone propionate, epitiostanol,mepitiostane, testolactone; anti-adrenals such as aminoglutethimide,mitotane, trilostane; folic acid replenisher such as frolinic acid;aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil;amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine;diaziquone; elfomithine; elliptinium acetate; an epothilone; etoglucid;gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids suchas maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidamnol;nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone;podophyllinic acid; 2-ethylhydrazide; procarbazine; PSK® polysaccharidecomplex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin;sizofuran; spirogermanium; tenuazonic acid; triaziquone;2,2′,2″-trichlorotriethylamine; trichothecenes (especially T-2 toxin,verracurin A, roridin A and anguidine); urethan; vindesine; dacarbazine;mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine;arabinoside (“Ara-C”); cyclophosphamide; thiotepa; taxoids, e.g., TAXOL(paclitaxel; Bristol-Myers Squibb Oncology, Princeton, N.J.), ABRAXANE®(Cremophor-free), albumin-engineered nanoparticle formulations ofpaclitaxel (American Pharmaceutical Partners, Schaumberg, Ill.), andTAXOTERE® (docetaxel, doxetaxel; Sanofi-Aventis); chlorambucil; GEMZAR®(gemcitabine); 6-thioguanine; mercaptopurine; methotrexate; platinumanalogs such as cisplatin and carboplatin; vinblastine; etoposide(VP-16); ifosfamide; mitoxantrone; vincristine; NAVELBINE®(vinorelbine); novantrone; teniposide; edatrexate; daunomycin;aminopterin; capecitabine (XELODA®); ibandronate; CPT-11; topoisomeraseinhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such asretinoic acid; and pharmaceutically acceptable salts, acids andderivatives of any of the above.

Chemotherapeutic agent also includes (i) anti-hormonal agents that actto regulate or inhibit hormone action on tumors such as anti-estrogensand selective estrogen receptor modulators (SERMs), including, forexample, tamoxifen (including NOLVADEX®; tamoxifen citrate), raloxifene,droloxifene, iodoxyfene, 4-hydroxytamoxifen, trioxifene, keoxifene,LY117018, onapristone, and FARESTON® (toremifine citrate); (ii)aromatase inhibitors that inhibit the enzyme aromatase, which regulatesestrogen production in the adrenal glands, such as, for example,4(5)-imidazoles, aminoglutethimide, MEGASE® (megestrol acetate),AROMASIN® (exemestane; Pfizer), formestanie, fadrozole, RIVISOR®(vorozole), FEMARA® (letrozole; Novartis), and ARIMIDEX® (anastrozole;Astra7eneca); (iii) anti-androgens such as flutamide, nilutamide,bicalutamide, leuprolide and goserelin; buserelin, tripterelin,medroxyprogesterone acetate, diethylstilbestrol, premarin,fluoxymesterone, all transretionic acid, fenretinide, as well astroxacitabine (a 1,3-dioxolane nucleoside cytosine analog); (iv) proteinkinase inhibitors; (v) lipid kinase inhibitors; (vi) antisenseoligonucleotides, particularly those which inhibit expression of genesin signaling pathways implicated in aberrant cell proliferation, suchas, for example, PKC-alpha, Ralf and H-Ras; (vii) ribozymes such as VEGFexpression inhibitors (e.g., ANGIOZYME®) and HER2 expression inhibitors;(viii) vaccines such as gene therapy vaccines, for example, ALLOVECTIN®,LEUVECTIN®, and VAXID®; PROLEUKIN®, rIL-2; a topoisomerase 1 inhibitorsuch as LURTOTECAN®; ABARELIX® rmRH; and (ix) pharmaceuticallyacceptable salts, acids and derivatives of any of the above.

Chemotherapeutic agent also includes antibodies such as alemtuzumab(Campath), bevacizumab (AVASTIN®, Genentech); cetuximab (ERBITUX®,Imclone); panitumumab (VECTIBIX®, Amgen), rituximab (RITUXAN®,Genentech/Biogen Idec), pertuzumab (OMNITARG®, 2C4, Genentech),trastuzumab (HERCEPTIN®, Genentech), tositumomab (Bexxar, Corixia), andthe antibody drug conjugate, gemtuzumab ozogamicin (MYLOTARG®, Wyeth).Additional humanized monoclonal antibodies with therapeutic potential asagents in combination with the compounds of the invention include:apolizumab, aselizumab, atlizumab, bapineuzumab, bivatuzumab mertansine,cantuzumab mertansine, cedelizumab, certolizumab pegol, cidfusituzumab,cidtuzumab, daclizumab, eculizumab, efalizumab, epratuzumab, erlizumab,felvizumab, fontolizumab, gemtuzumab ozogamicin, inotuzumab ozogamicin,ipilimumab, labetuzumab, lintuzumab, matuzumab, mepolizumab,motavizumab, motovizumab, natalizumab, nimotuzumab, nolovizumab,numavizumab, ocrelizumab, omalizumab, palivizumab, pascolizumab,pecfusituzumab, pectuzumab, pexelizumab, ralivizumab, ranibizumab,reslivizumab, reslizumab, resyvizumab, rovelizumab, ruplizumab,sibrotuzumab, siplizumab, sontuzumab, tacatuzumab tetraxetan,tadocizumab, talizumab, tefibazumab, tocilizumab, toralizumab,tucotuzumab celmoleukin, tucusituzumab, umavizumab, urtoxazumab,ustekinumab, visilizumab, and the anti-interleukin-12 (ABT-874/J695,Wyeth Research and Abbott Laboratories) which is a recombinantexclusively human-sequence, full-length IgG₁ λ antibody geneticallymodified to recognize interleukin-12 p40 protein.

Chemotherapeutic agent also includes “EGFR inhibitors,” which refers tocompounds that bind to or otherwise interact directly with EGFR andprevent or reduce its signaling activity, and is alternatively referredto as an “EGFR antagonist.” Examples of such agents include antibodiesand small molecules that bind to EGFR. Examples of antibodies which bindto EGFR include MAb 579 (ATCC CRL HB 8506), MAb 455 (ATCC CRL HB8507),MAb 225 (ATCC CRL 8508), MAb 528 (ATCC CRL 8509) (see, U.S. Pat. No.4,943,533, Mendelsohn et al.) and variants thereof, such as chimerized225 (C225 or Cetuximab; ERBUTIX) and reshaped human 225 (H225) (see, WO96/40210, Imclone Systems Inc.); IMC-11F8, a fully human, EGFR-targetedantibody (Imclone); antibodies that bind type II mutant EGFR (U.S. Pat.No. 5,212,290); humanized and chimeric antibodies that bind EGFR asdescribed in U.S. Pat. No. 5,891,996; and human antibodies that bindEGFR, such as ABX-EGF or Panitumumab (see WO98/50433, Abgenix/Amgen);EMD 55900 (Stragliotto et al. Eur. J. Cancer 32A:636-640 (1996));EMD7200 (matuzumab) a humanized EGFR antibody directed against EGFR thatcompetes with both EGF and TGF-alpha for EGFR binding (EMD/Merck); humanEGFR antibody, HuMax-EGFR (GenMab); fully human antibodies known asE1.1, E2.4, E2.5, E6.2, E6.4, E2.11, E6. 3 and E7.6. 3 and described inU.S. Pat. No. 6,235,883; MDX-447 (Medarex Inc); and mAb 806 or humanizedmAb 806 (Johns et al., J. Biol. Chem. 279(29):30375-30384 (2004)). Theanti-EGFR antibody may be conjugated with a cytotoxic agent, thusgenerating an immunoconjugate (see, e.g., EP659439A2, Merck PatentGmbH). EGFR antagonists include small molecules such as compoundsdescribed in U.S. Pat. Nos. 5,616,582, 5,457,105, 5,475,001, 5,654,307,5,679,683, 6,084,095, 6,265,410, 6,455,534, 6,521,620, 6,596,726,6,713,484, 5,770,599, 6,140,332, 5,866,572, 6,399,602, 6,344,459,6,602,863, 6,391,874, 6,344,455, 5,760,041, 6,002,008, and 5,747,498, aswell as the following PCT publications: WO98/14451, WO98/50038,WO99/09016, and WO99/24037. Particular small molecule EGFR antagonistsinclude OSI-774 (CP-358774, erlotinib, TARCEVA® Genentech/OSIPharmaceuticals); PD 183805 (CI 1033, 2-propenamide,N-[4-[(3-chloro-4-fluorophenyl)amino]-7-[3-(4-morpholinyl)propoxy]-6-quinazolinyl]-,dihydrochloride, Pfizer Inc.); ZD1839, gefitinib (IRESSA®)4-(3′-Chloro-4′-fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline,Astra7eneca); ZM 105180 ((6-amino-4-(3-methylphenyl-amino)-quinazoline,Zeneca); BIBX-1382(N8-(3-chloro-4-fluoro-phenyl)-N2-(1-methyl-piperidin-4-yl)-pyrimido[5,4-d]pyrimidine-2,8-diamine,Boehringer Ingelheim); PKI-166((R)-4-[4-[(1-phenylethyl)amino]-1H-pyrrolo[2,3-d]pyrimidin-6-yl]-phenol);(R)-6-(4-hydroxyphenyl)-4-[(1-phenylethyl)amino]-7H-pyrrolo[2,3-d]pyrimidine);CL-387785 (N-[4-[(3-bromophenyl)amino]-6-quinazolinyl]-2-butynamide);EKB-569(N-[4-[(3-chloro-4-fluorophenyl)amino]-3-cyano-7-ethoxy-6-quinolinyl]-4-(dimethylamino)-2-butenamide)(Wyeth); AG1478 (Pfizer); AG1571 (SU 5271; Pfizer); dual EGFR/HER2tyrosine kinase inhibitors such as lapatinib (TYKERB®, GSK572016 orN-[3-chloro-4-[(3fluorophenyl)methoxy]phenyl]-6[5[[[2methylsulfonyl)ethyl]amino]methyl]-2-furanyl]-4-quinazolinamine).

Chemotherapeutic agents also include “tyrosine kinase inhibitors”including the EGFR-targeted drugs noted in the preceding paragraph;small molecule HER2 tyrosine kinase inhibitor such as TAK165 availablefrom Takeda; CP-724,714, an oral selective inhibitor of the ErbB2receptor tyrosine kinase (Pfizer and OSI); dual-HER inhibitors such asEKB-569 (available from Wyeth) which preferentially binds EGFR butinhibits both HER2 and EGFR-overexpressing cells; lapatinib (GSK572016;available from Glaxo-SmithKline), an oral HER2 and EGFR tyrosine kinaseinhibitor; PKI-166 (available from Novartis); pan-HER inhibitors such ascanertinib (CI-1033; Pharmacia); Raf-1 inhibitors such as antisenseagent ISIS-5132 available from ISIS Pharmaceuticals which inhibit Raf-1signaling; non-HER targeted TK inhibitors such as imatinib mesylate(GLEEVEC®, available from Glaxo SmithKline); multi-targeted tyrosinekinase inhibitors such as sunitinib (SUTENT®, available from Pfizer);VEGF receptor tyrosine kinase inhibitors such as vatalanib(PTK787/ZK222584, available from Novartis/Schering AG); MAPKextracellular regulated kinase I inhibitor CI-1040 (available fromPharmacia); quinazolines, such as PD 153035,4-(3-chloroanilino)quinazoline; pyridopyrimidines; pyrimidopyrimidines; pyrrolopyrimidines,such as CGP 59326, CGP 60261 and CGP 62706; pyrazolopyrimidines,4-(phenylamino)-7H-pyrrolo[2,3-d] pyrimidines; curcumin (diferuloylmethane, 4,5-bis (4-fluoroanilino)phthalimide); tyrphostines containingnitrothiophene moieties; PD-0183805 (Warner-Lamber); antisense molecules(e.g. those that bind to HER-encoding nucleic acid); quinoxalines (U.S.Pat. No. 5,804,396); tryphostins (U.S. Pat. No. 5,804,396); ZD6474(Astra Zeneca); PTK-787 (Novartis/Schering AG); pan-HER inhibitors suchas CI-1033 (Pfizer); Affinitac (ISIS 3521; Isis/Lilly); imatinibmesylate (GLEEVEC®); PKI 166 (Novartis); GW2016 (Glaxo SmithKline);CI-1033 (Pfizer); EKB-569 (Wyeth); Semaxinib (Pfizer); ZD6474(AstraZeneca); PTK-787 (Novartis/Schering AG); INC-1C11 (Imclone),rapamycin (sirolimus, RAPAMUNE®); or as described in any of thefollowing patent publications: U.S. Pat. No. 5,804,396; WO 1999/09016(American Cyanamid); WO 1998/43960 (American Cyanamid); WO 1997/38983(Warner Lambert); WO 1999/06378 (Warner Lambert); WO 1999/06396 (WarnerLambert); WO 1996/30347 (Pfizer, Inc); WO 1996/33978 (Zeneca); WO1996/3397 (Zeneca) and WO 1996/33980 (Zeneca).

Chemotherapeutic agents also include dexamethasone, interferons,colchicine, metoprine, cyclosporine, amphotericin, metronidazole,alemtuzumab, alitretinoin, allopurinol, amifostine, arsenic trioxide,asparaginase, BCG live, bevacuzimab, bexarotene, cladribine,clofarabine, darbepoetin alfa, denileukin, dexrazoxane, epoetin alfa,elotinib, filgrastim, histrelin acetate, ibritumomab, interferonalfa-2a, interferon alfa-2b, lenalidomide, levamisole, mesna,methoxsalen, nandrolone, nelarabine, nofetumomab, oprelvekin,palifermin, pamidronate, pegademase, pegaspargase, pegfilgrastim,pemetrexed disodium, plicamycin, porfimer sodium, quinacrine,rasburicase, sargramostim, temozolomide, VM-26, 6-TG, toremifene,tretinoin, ATRA, valrubicin, zoledronate, and zoledronic acid, andpharmaceutically acceptable salts thereof.

Chemotherapeutic agents also include hydrocortisone, hydrocortisoneacetate, cortisone acetate, tixocortol pivalate, triamcinoloneacetonide, triamcinolone alcohol, mometasone, amcinonide, budesonide,desonide, fluocinonide, fluocinolone acetonide, betamethasone,betamethasone sodium phosphate, dexamethasone, dexamethasone sodiumphosphate, fluocortolone, hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate and fluprednidene acetate; immuneselective anti-inflammatory peptides (ImSAIDs) such asphenylalanine-glutamine-glycine (FEG) and its D-isomeric form (feG)(IMULAN BioTherapeutics, LLC); anti-rheumatic drugs such asazathioprine, ciclosporin (cyclosporine A), D-penicillamine, gold salts,hydroxychloroquine, leflunomideminocycline, sulfasalazine, tumornecrosis factor alpha (TNFα) blockers such as etanercept (Enbrel),infliximab (Remicade), adalimumab (Humira), certolizumab pegol (Cimzia),golimumab (Simponi), Interleukin 1 (IL-1) blockers such as anakinra(Kineret), T cell costimulation blockers such as abatacept (Orencia),Interleukin 6 (IL-6) blockers such as tocilizumab (ACTEMRA®);Interleukin 13 (IL-13) blockers such as lebrikizumab; Interferon alpha(IFN) blockers such as Rontalizumab; Beta 7 integrin blockers such asrhuMAb Beta7; IgE pathway blockers such as Anti-M1 prime; Secretedhomotrimeric LTa3 and membrane bound heterotrimer LTa1/β2 blockers suchas Anti-lymphotoxin alpha (LTa); radioactive isotopes (e.g., At²¹¹,I¹³¹, I¹²⁵, Y⁹⁰, Re¹⁸⁶, Re¹⁸⁸, Sm¹⁵³, Bi²¹², P³², Pb²¹² and radioactiveisotopes of Lu); miscellaneous investigational agents such asthioplatin, PS-341, phenylbutyrate, ET-18-OCH3, or farnesyl transferaseinhibitors (L-739749, L-744832); polyphenols such as quercetin,resveratrol, piceatannol, epigallocatechine gallate, theaflavins,flavanols, procyanidins, betulinic acid and derivatives thereof;autophagy inhibitors such as chloroquine; delta-9-tetrahydrocannabinol(dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinicacid; acetylcamptothecin, scopolectin, and 9-aminocamptothecin);podophyllotoxin; tegafur (UFTORAL®); bexarotene (TARGRETIN®);bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®),etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®),alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), orrisedronate (ACTONEL®); and epidermal growth factor receptor (EGF-R);vaccines such as THERATOPE® vaccine; perifosine, COX-2 inhibitor (e.g.celecoxib or etoricoxib), proteosome inhibitor (e.g. PS341); CCI-779;tipifarnib (R11577); orafenib, ABT510; Bc1-2 inhibitor such asoblimersen sodium (GENASENSE®); pixantrone; farnesyltransferaseinhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceuticallyacceptable salts, acids or derivatives of any of the above; as well ascombinations of two or more of the above such as CHOP, an abbreviationfor a combined therapy of cyclophosphamide, doxorubicin, vincristine,and prednisolone; and FOLFOX, an abbreviation for a treatment regimenwith oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin.

Chemotherapeutic agents also include non-steroidal anti-inflammatorydrugswith analgesic, antipyretic and anti-inflammatory effects. NSAIDsinclude non-selective inhibitors of the enzyme cyclooxygenase. Specificexamples of NSAIDs include aspirin, propionic acid derivatives such asibuprofen, fenoprofen, ketoprofen, flurbiprofen, oxaprozin and naproxen,acetic acid derivatives such as indomethacin, sulindac, etodolac,diclofenac, enolic acid derivatives such as piroxicam, meloxicam,tenoxicam, droxicam, lornoxicam and isoxicam, fenamic acid derivativessuch as mefenamic acid, meclofenamic acid, flufenamic acid, tolfenamicacid, and COX-2 inhibitors such as celecoxib, etoricoxib, lumiracoxib,parecoxib, rofecoxib, rofecoxib, and valdecoxib. NSAIDs can be indicatedfor the symptomatic relief of conditions such as rheumatoid arthritis,osteoarthritis, inflammatory arthropathies, ankylosing spondylitis,psoriatic arthritis, Reiter's syndrome, acute gout, dysmenorrhoea,metastatic bone pain, headache and migraine, postoperative pain,mild-to-moderate pain due to inflammation and tissue injury, pyrexia,ileus, and renal colic.

A “growth inhibitory agent” when used herein refers to a compound orcomposition which inhibits growth of a cell either in vitro or in vivo.In one embodiment, growth inhibitory agent is growth inhibitory antibodythat prevents or reduces proliferation of a cell expressing an antigento which the antibody binds. In another embodiment, the growthinhibitory agent may be one which significantly reduces the percentageof cells in S phase. Examples of growth inhibitory agents include agentsthat block cell cycle progression (at a place other than S phase), suchas agents that induce G1 arrest and M-phase arrest. Classical M-phaseblockers include the vincas (vincristine and vinblastine), taxanes, andtopoisomerase II inhibitors such as doxorubicin, epirubicin,daunorubicin, etoposide, and bleomycin. Those agents that arrest G1 alsospill over into S-phase arrest, for example, DNA alkylating agents suchas tamoxifen, prednisone, dacarbazine, mechlorethamine, cisplatin,methotrexate, 5-fluorouracil, and ara-C. Further information can befound in Mendelsohn and Israel, eds., The Molecular Basis of Cancer,Chapter 1, entitled “Cell cycle regulation, oncogenes, andantineoplastic drugs” by Murakami et al. (W.B. Saunders, Philadelphia,1995), e.g., p. 13. The taxanes (paclitaxel and docetaxel) areanticancer drugs both derived from the yew tree. Docetaxel (TAXOTERE®,Rhone-Poulenc Rorer), derived from the European yew, is a semisyntheticanalogue of paclitaxel (TAXOL®, Bristol-Myers Squibb). Paclitaxel anddocetaxel promote the assembly of microtubules from tubulin dimers andstabilize microtubules by preventing depolymerization, which results inthe inhibition of mitosis in cells.

By “radiation therapy” is meant the use of directed gamma rays or betarays to induce sufficient damage to a cell so as to limit its ability tofunction normally or to destroy the cell altogether. It will beappreciated that there will be many ways known in the art to determinethe dosage and duration of treatment. Typical treatments are given as aone-time administration and typical dosages range from 10 to 200 units(Grays) per day.

A “subject” or an “individual” for purposes of treatment refers to anyanimal classified as a mammal, including humans, domestic and farmanimals, and zoo, sports, or pet animals, such as dogs, horses, cats,cows, etc. Preferably, the mammal is human. An individual or subject maybe a patient.

The term “antibody” herein is used in the broadest sense andspecifically covers monoclonal antibodies (including full lengthmonoclonal antibodies), polyclonal antibodies, multispecific antibodies(e.g., bispecific antibodies), and antibody fragments so long as theyexhibit the desired biological activity.

An “isolated” antibody is one which has been identified and separatedand/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with research, diagnostic or therapeutic uses for theantibody, and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In some embodiments, an antibody is purified(1) to greater than 95% by weight of antibody as determined by, forexample, the Lowry method, and in some embodiments, to greater than 99%by weight; (2) to a degree sufficient to obtain at least 15 residues ofN-terminal or internal amino acid sequence by use of, for example, aspinning cup sequenator, or (3) to homogeneity by SDS-PAGE underreducing or nonreducing conditions using, for example, Coomassie blue orsilver stain. Isolated antibody includes the antibody in situ withinrecombinant cells since at least one component of the antibody's naturalenvironment will not be present. Ordinarily, however, isolated antibodywill be prepared by at least one purification step.

“Native antibodies” are usually heterotetrameric glycoproteins of about150,000 daltons, composed of two identical light (L) chains and twoidentical heavy (H) chains. Each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries among the heavy chains of different immunoglobulin isotypes. Eachheavy and light chain also has regularly spaced intrachain disulfidebridges. Each heavy chain has at one end a variable domain (V_(H))followed by a number of constant domains. Each light chain has avariable domain at one end (V_(L)) and a constant domain at its otherend; the constant domain of the light chain is aligned with the firstconstant domain of the heavy chain, and the light chain variable domainis aligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light chainand heavy chain variable domains.

The term “constant domain” refers to the portion of an immunoglobulinmolecule having a more conserved amino acid sequence relative to theother portion of the immunoglobulin, the variable domain, which containsthe antigen binding site. The constant domain contains the C_(H)1,C_(H)2 and C_(H)3 domains (collectively, CH) of the heavy chain and theCHL (or CL) domain of the light chain.

The “variable region” or “variable domain” of an antibody refers to theamino-terminal domains of the heavy or light chain of the antibody. Thevariable domain of the heavy chain may be referred to as “V_(H).” Thevariable domain of the light chain may be referred to as “V_(L).” Thesedomains are generally the most variable parts of an antibody and containthe antigen-binding sites.

The term “variable” refers to the fact that certain portions of thevariable domains differ extensively in sequence among antibodies and areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions (HVRs) both in thelight-chain and the heavy-chain variable domains. The more highlyconserved portions of variable domains are called the framework regions(FR). The variable domains of native heavy and light chains eachcomprise four FR regions, largely adopting a beta-sheet configuration,connected by three HVRs, which form loops connecting, and in some casesforming part of, the beta-sheet structure. The HVRs in each chain areheld together in close proximity by the FR regions and, with the HVRsfrom the other chain, contribute to the formation of the antigen-bindingsite of antibodies (see Kabat et al., Sequences of Proteins ofImmunological Interest, Fifth Edition, National Institute of Health,Bethesda, Md. (1991)). The constant domains are not involved directly inthe binding of an antibody to an antigen, but exhibit various effectorfunctions, such as participation of the antibody in antibody-dependentcellular toxicity.

The “light chains” of antibodies (immunoglobulins) from any mammalianspecies can be assigned to one of two clearly distinct types, calledkappa (“κ”) and lambda (“λ”), based on the amino acid sequences of theirconstant domains.

The term IgG “isotype” or “subclass” as used herein is meant any of thesubclasses of immunoglobulins defined by the chemical and antigeniccharacteristics of their constant regions.

Depending on the amino acid sequences of the constant domains of theirheavy chains, antibodies (immunoglobulins) can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG₁, IgG₂, IgG₃, IgG₄, IgA₁, and IgA₂. Theheavy chain constant domains that correspond to the different classes ofimmunoglobulins are called α, γ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known and described generally in, for example,Abbas et al. Cellular and Mol. Immunology, 4th ed. (W.B. Saunders, Co.,2000). An antibody may be part of a larger fusion molecule, formed bycovalent or non-covalent association of the antibody with one or moreother proteins or peptides.

The terms “full length antibody,” “intact antibody” and “whole antibody”are used herein interchangeably to refer to an antibody in itssubstantially intact form, not antibody fragments as defined below. Theterms particularly refer to an antibody with heavy chains that containan Fc region.

A “naked antibody” for the purposes herein is an antibody that is notconjugated to a cytotoxic moiety or radiolabel.

“Antibody fragments” comprise a portion of an intact antibody,preferably comprising the antigen binding region thereof. In someembodiments, the antibody fragment described herein is anantigen-binding fragment. Examples of antibody fragments include Fab,Fab′, F(ab′)₂, and Fv fragments; diabodies; linear antibodies;single-chain antibody molecules; and multispecific antibodies formedfrom antibody fragments.

Papain digestion of antibodies produces two identical antigen-bindingfragments, called “Fab” fragments, each with a single antigen-bindingsite, and a residual “Fc” fragment, whose name reflects its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen-combining sites and is still capable of cross-linkingantigen.

“Fv” is the minimum antibody fragment which contains a completeantigen-binding site. In one embodiment, a two-chain Fv species consistsof a dimer of one heavy- and one light-chain variable domain in tight,non-covalent association. In a single-chain Fv (scFv) species, oneheavy- and one light-chain variable domain can be covalently linked by aflexible peptide linker such that the light and heavy chains canassociate in a “dimeric” structure analogous to that in a two-chain Fvspecies. It is in this configuration that the three HVRs of eachvariable domain interact to define an antigen-binding site on thesurface of the VH-VL dimer. Collectively, the six HVRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three HVRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

The Fab fragment contains the heavy- and light-chain variable domainsand also contains the constant domain of the light chain and the firstconstant domain (CH1) of the heavy chain. Fab′ fragments differ from Fabfragments by the addition of a few residues at the carboxy terminus ofthe heavy chain CH1 domain including one or more cysteines from theantibody hinge region. Fab′-SH is the designation herein for Fab′ inwhich the cysteine residue(s) of the constant domains bear a free thiolgroup. F(ab′)2 antibody fragments originally were produced as pairs ofFab′ fragments which have hinge cysteines between them. Other chemicalcouplings of antibody fragments are also known.

“Single-chain Fv” or “scFv” antibody fragments comprise the VH and VLdomains of antibody, wherein these domains are present in a singlepolypeptide chain. Generally, the scFv polypeptide further comprises apolypeptide linker between the VH and VL domains which enables the scFvto form the desired structure for antigen binding. For a review of scFv,see, e.g., Plückthun, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds., (Springer-Verlag, New York, 1994), pp.269-315.

The term “diabodies” refers to antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (VH) connected to a light-chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies may be bivalent orbispecific. Diabodies are described more fully in, for example, EP404,097; WO 1993/01161; Hudson et al., Nat. Med. 9:129-134 (2003); andHollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993).Triabodies and tetrabodies are also described in Hudson et al., Nat.Med. 9:129-134 (2003).

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,e.g., the individual antibodies comprising the population are identicalexcept for possible mutations, e.g., naturally occurring mutations, thatmay be present in minor amounts. Thus, the modifier “monoclonal”indicates the character of the antibody as not being a mixture ofdiscrete antibodies. In certain embodiments, such a monoclonal antibodytypically includes an antibody comprising a polypeptide sequence thatbinds a target, wherein the target-binding polypeptide sequence wasobtained by a process that includes the selection of a single targetbinding polypeptide sequence from a plurality of polypeptide sequences.For example, the selection process can be the selection of a uniqueclone from a plurality of clones, such as a pool of hybridoma clones,phage clones, or recombinant DNA clones. It should be understood that aselected target binding sequence can be further altered, for example, toimprove affinity for the target, to humanize the target bindingsequence, to improve its production in cell culture, to reduce itsimmunogenicity in vivo, to create a multispecific antibody, etc., andthat an antibody comprising the altered target binding sequence is alsoa monoclonal antibody of this invention. In contrast to polyclonalantibody preparations, which typically include different antibodiesdirected against different determinants (epitopes), each monoclonalantibody of a monoclonal antibody preparation is directed against asingle determinant on an antigen. In addition to their specificity,monoclonal antibody preparations are advantageous in that they aretypically uncontaminated by other immunoglobulins.

The modifier “monoclonal” indicates the character of the antibody asbeing obtained from a substantially homogeneous population ofantibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with the invention may be made by avariety of techniques, including, for example, the hybridoma method(e.g., Kohler and Milstein, Nature, 256:495-97 (1975); Hongo et al.,Hybridoma, 14 (3): 253-260 (1995), Harlow et al., Antibodies: ALaboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988);Hammerling et al., in: Monoclonal Antibodies and T-Cell Hybridomas563-681 (Elsevier, N.Y., 1981)), recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567), phage-display technologies (see, e.g.,Clackson et al., Nature, 352: 624-628 (1991); Marks et al., J. Mol.Biol. 222: 581-597 (1992); Sidhu et al., J. Mol. Biol. 338(2): 299-310(2004); Lee et al., J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse,Proc. Natl. Acad. Sci. USA 101(34): 12467-12472 (2004); and Lee et al.,J. Immunol. Methods 284(1-2): 119-132 (2004), and technologies forproducing human or human-like antibodies in animals that have parts orall of the human immunoglobulin loci or genes encoding humanimmunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO1996/33735; WO 1991/10741; Jakobovits et al., Proc. Natl. Acad. Sci. USA90: 2551 (1993); Jakobovits et al., Nature 362: 255-258 (1993);Bruggemann et al., Year in Immunol. 7:33 (1993); U.S. Pat. Nos.5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and U.S. Pat. No.5,661,016; Marks et al., Bio/Technology 10: 779-783 (1992); Lonberg etal., Nature 368: 856-859 (1994); Morrison, Nature 368: 812-813 (1994);Fishwild et al., Nature Biotechnol. 14: 845-851 (1996); Neuberger,Nature Biotechnol. 14: 826 (1996); and Lonberg and Huszar, Intern. Rev.Immunol. 13: 65-93 (1995).

The monoclonal antibodies herein specifically include “chimeric”antibodies in which a portion of the heavy and/or light chain isidentical with or homologous to corresponding sequences in antibodiesderived from a particular species or belonging to a particular antibodyclass or subclass, while the remainder of the chain(s) is identical withor homologous to corresponding sequences in antibodies derived fromanother species or belonging to another antibody class or subclass, aswell as fragments of such antibodies, so long as they exhibit thedesired biological activity (see, e.g., U.S. Pat. No. 4,816,567; andMorrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)).Chimeric antibodies include PRIMATIZED® antibodies wherein theantigen-binding region of the antibody is derived from an antibodyproduced by, e.g., immunizing macaque monkeys with the antigen ofinterest.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies that contain minimal sequence derived from non-humanimmunoglobulin. In one embodiment, a humanized antibody is a humanimmunoglobulin (recipient antibody) in which residues from a HVR of therecipient are replaced by residues from a HVR of a non-human species(donor antibody) such as mouse, rat, rabbit, or nonhuman primate havingthe desired specificity, affinity, and/or capacity. In some instances,FR residues of the human immunoglobulin are replaced by correspondingnon-human residues. Furthermore, humanized antibodies may compriseresidues that are not found in the recipient antibody or in the donorantibody. These modifications may be made to further refine antibodyperformance. In general, a humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the hypervariable loops correspondto those of a non-human immunoglobulin, and all or substantially all ofthe FRs are those of a human immunoglobulin sequence. The humanizedantibody optionally will also comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see, e.g., Jones et al., Nature321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); andPresta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g.,Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998);Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross,Curr. Op. Biotech. 5:428-433 (1994); and U.S. Pat. Nos. 6,982,321 and7,087,409.

A “human antibody” is one which possesses an amino acid sequence whichcorresponds to that of an antibody produced by a human and/or has beenmade using any of the techniques for making human antibodies asdisclosed herein. This definition of a human antibody specificallyexcludes a humanized antibody comprising non-human antigen-bindingresidues. Human antibodies can be produced using various techniquesknown in the art, including phage-display libraries. Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991). Also available for the preparation of human monoclonalantibodies are methods described in Cole et al., Monoclonal Antibodiesand Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991). See also van Dijk and van de Winkel,Curr. Opin. Pharmacol., 5: 368-74 (2001). Human antibodies can beprepared by administering the antigen to a transgenic animal that hasbeen modified to produce such antibodies in response to antigenicchallenge, but whose endogenous loci have been disabled, e.g., immunizedxenomice (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 regardingXENOMOUSE™ technology). See also, for example, Li et al., Proc. Natl.Acad. Sci. USA, 103:3557-3562 (2006) regarding human antibodiesgenerated via a human B-cell hybridoma technology.

A “species-dependent antibody” is one which has a stronger bindingaffinity for an antigen from a first mammalian species than it has for ahomologue of that antigen from a second mammalian species. Normally, thespecies-dependent antibody “binds specifically” to a human antigen(e.g., has a binding affinity (K_(D)) value of no more than about 1×10⁻⁷M, preferably no more than about 1×10⁻⁸ M and preferably no more thanabout 1×10⁻⁹ M) but has a binding affinity for a homologue of theantigen from a second nonhuman mammalian species which is at least about50 fold, or at least about 500 fold, or at least about 1000 fold, weakerthan its binding affinity for the human antigen. The species-dependentantibody can be any of the various types of antibodies as defined above,but preferably is a humanized or human antibody.

The term “hypervariable region,” “HVR,” or “HV,” when used herein refersto the regions of an antibody variable domain which are hypervariable insequence and/or form structurally defined loops. Generally, antibodiescomprise six HVRs; three in the VH (H1, H2, H3), and three in the VL(L1, L2, L3). In native antibodies, H3 and L3 display the most diversityof the six HVRs, and H3 in particular is believed to play a unique rolein conferring fine specificity to antibodies. See, e.g., Xu et al.,Immunity 13:37-45 (2000); Johnson and Wu, in Methods in MolecularBiology 248:1-25 (Lo, ed., Human Press, Totowa, N.J., 2003). Indeed,naturally occurring camelid antibodies consisting of a heavy chain onlyare functional and stable in the absence of light chain. See, e.g.,Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al.,Nature Struct. Biol. 3:733-736 (1996).

A number of HVR delineations are in use and are encompassed herein. TheKabat Complementarity Determining Regions (CDRs) are based on sequencevariability and are the most commonly used (Kabat et al., Sequences ofProteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)). Chothia refersinstead to the location of the structural loops (Chothia and Lesk J.Mol. Biol. 196:901-917 (1987)). The AbM HVRs represent a compromisebetween the Kabat HVRs and Chothia structural loops, and are used byOxford Molecular's AbM antibody modeling software. The “contact” HVRsare based on an analysis of the available complex crystal structures.The residues from each of these HVRs are noted below.

Loop Kabat AbM Chothia Contact L1 L24-L34 L24-L34 L26-L32 L30-L36 L2L50-L56 L50-L56 L50-L52 L46-L55 L3 L89-L97 L89-L97 L91-L96 L89-L96 H1H31-H35B H26-H35B H26-H32 H30-H35B (Kabat Numbering) H1 H31-H35 H26-H35H26-H32 H30-H35 (Chothia Numbering) H2 H50-H65 H50-H58 H53-H55 H47-H58H3 H95-H102 H95-H102 H96-H101 H93-H101

HVRs may comprise “extended HVRs” as follows: 24-36 or 24-34 (L1), 46-56or 50-56 (L2) and 89-97 or 89-96 (L3) in the VL and 26-35 (H1), 50-65 or49-65 (H2) and 93-102, 94-102, or 95-102 (H3) in the VH. The variabledomain residues are numbered according to Kabat et al., supra, for eachof these definitions.

“Framework” or “FR” residues are those variable domain residues otherthan the HVR residues as herein defined.

The term “variable domain residue numbering as in Kabat” or “amino acidposition numbering as in Kabat,” and variations thereof, refers to thenumbering system used for heavy chain variable domains or light chainvariable domains of the compilation of antibodies in Kabat et al.,supra. Using this numbering system, the actual linear amino acidsequence may contain fewer or additional amino acids corresponding to ashortening of, or insertion into, a FR or HVR of the variable domain.For example, a heavy chain variable domain may include a single aminoacid insert (residue 52a according to Kabat) after residue 52 of H2 andinserted residues (e.g. residues 82a, 82b, and 82c, etc. according toKabat) after heavy chain FR residue 82. The Kabat numbering of residuesmay be determined for a given antibody by alignment at regions ofhomology of the sequence of the antibody with a “standard” Kabatnumbered sequence.

The Kabat numbering system is generally used when referring to a residuein the variable domain (approximately residues 1-107 of the light chainand residues 1-113 of the heavy chain) (e.g., Kabat et al., Sequences ofImmunological Interest. 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, Md. (1991)). The “EU numbering system”or “EU index” is generally used when referring to a residue in animmunoglobulin heavy chain constant region (e.g., the EU index reportedin Kabat et al., supra). The “EU index as in Kabat” refers to theresidue numbering of the human IgG1 EU antibody.

The expression “linear antibodies” refers to the antibodies described inZapata et al. (1995 Protein Eng, 8(10):1057-1062). Briefly, theseantibodies comprise a pair of tandem Fd segments (VH-CH1-VH-CH1) which,together with complementary light chain polypeptides, form a pair ofantigen binding regions. Linear antibodies can be bispecific ormonospecific.

As use herein, the term “binds”, “specifically binds to” or is “specificfor” refers to measurable and reproducible interactions such as bindingbetween a target and an antibody, which is determinative of the presenceof the target in the presence of a heterogeneous population of moleculesincluding biological molecules. For example, an antibody that binds toor specifically binds to a target (which can be an epitope) is anantibody that binds this target with greater affinity, avidity, morereadily, and/or with greater duration than it binds to other targets. Inone embodiment, the extent of binding of an antibody to an unrelatedtarget is less than about 10% of the binding of the antibody to thetarget as measured, e.g., by a radioimmunoassay (RIA). In certainembodiments, an antibody that specifically binds to a target has adissociation constant (Kd) of ≤1 μM, ≤100 nM, ≤10 nM, ≤1 nM, or ≤0.1 nM.In certain embodiments, an antibody specifically binds to an epitope ona protein that is conserved among the protein from different species. Inanother embodiment, specific binding can include, but does not requireexclusive binding.

The term “bispecific” means that the antigen binding molecule is able tospecifically bind to at least two distinct antigenic determinants.Typically, a bispecific antigen binding molecule comprises two antigenbinding sites, each of which is specific for a different antigenicdeterminant. In certain embodiments the bispecific antigen bindingmolecule is capable of simultaneously binding two antigenicdeterminants, particularly two antigenic determinants expressed on twodistinct cells.

The term “antigen binding domain” refers to the part of an antibody thatcomprises the area which specifically binds to and is complementary topart or all of an antigen. An antigen binding domain may be provided by,for example, one or more antibody variable domains (also called antibodyvariable regions). Preferably, an antigen binding domain comprises anantibody light chain variable region (VL) and an antibody heavy chainvariable region (VH).

A “Fab molecule” refers to a protein consisting of the VH and CH1 domainof the heavy chain (the “Fab heavy chain”) and the VL and CL domain ofthe light chain (the “Fab light chain”) of an immunoglobulin.

By a “crossover” Fab molecule (also termed “Crossfab”) is meant a Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other), i.e.the crossover Fab molecule comprises a peptide chain composed of thelight chain variable domain VL and the heavy chain constant domain 1 CH1(VL-CH1, in N- to C-terminal direction), and a peptide chain composed ofthe heavy chain variable domain VH and the light chain constant domainCL (VH-CL, in N- to C-terminal direction). For clarity, in a crossoverFab molecule wherein the variable domains of the Fab light chain and theFab heavy chain are exchanged, the peptide chain comprising the heavychain constant domain 1 CH1 is referred to herein as the “heavy chain”of the (crossover) Fab molecule. Conversely, in a crossover Fab moleculewherein the constant domains of the Fab light chain and the Fab heavychain are exchanged, the peptide chain comprising the heavy chainvariable domain VH is referred to herein as the “heavy chain” of the(crossover) Fab molecule.

In contrast thereto, by a “conventional” Fab molecule is meant a Fabmolecule in its natural format, i.e. comprising a heavy chain composedof the heavy chain variable and constant domains (VH-CH1, in N- toC-terminal direction), and a light chain composed of the light chainvariable and constant domains (VL-CL, in N- to C-terminal direction).

The term “Fc domain” or “Fc region” herein is used to define aC-terminal region of an immunoglobulin heavy chain that contains atleast a portion of the constant region. The term includes nativesequence Fc regions and variant Fc regions. Although the boundaries ofthe Fc region of an IgG heavy chain might vary slightly, the human IgGheavy chain Fc region is usually defined to extend from Cys226, or fromPro230, to the carboxyl-terminus of the heavy chain. However, antibodiesproduced by host cells may undergo post-translational cleavage of one ormore, particularly one or two, amino acids from the C-terminus of theheavy chain. Therefore an antibody produced by a host cell by expressionof a specific nucleic acid molecule encoding a full-length heavy chainmay include the full-length heavy chain, or it may include a cleavedvariant of the full-length heavy chain (also referred to herein as a“cleaved variant heavy chain”). This may be the case where the final twoC-terminal amino acids of the heavy chain are glycine (G446) and lysine(K447, EU numbering). Therefore, the C-terminal lysine (Lys447), or theC-terminal glycine (Gly446) and lysine (K447), of the Fc region may ormay not be present. Unless otherwise specified herein, numbering ofamino acid residues in the Fc region or constant region is according tothe EU numbering system, also called the EU index, as described in Kabatet al., Sequences of Proteins of Immunological Interest, 5th Ed. PublicHealth Service, National Institutes of Health, Bethesda, Md., 1991 (seealso above). A “subunit” of an Fc domain as used herein refers to one ofthe two polypeptides forming the dimeric Fc domain, i.e. a polypeptidecomprising C-terminal constant regions of an immunoglobulin heavy chain,capable of stable self-association. For example, a subunit of an IgG Fcdomain comprises an IgG CH2 and an IgG CH3 constant domain.

By “fused” is meant that the components (e.g. a Fab molecule and an Fcdomain subunit) are linked by peptide bonds, either directly or via oneor more peptide linkers.

A “modification promoting heterodimerization” is a manipulation of thepeptide backbone or the post-translational modifications of apolypeptide, e.g. an immunoglobulin heavy chain, that reduces orprevents the association of the polypeptide with an identicalpolypeptide to form a homodimer. A modification promotingheterodimerization as used herein particularly includes separatemodifications made to each of two polypeptides desired to form a dimer,wherein the modifications are complementary to each other so as topromote association of the two polypeptides. For example, a modificationpromoting heterodimerization may alter the structure or charge of one orboth of the polypeptides desired to form a dimer so as to make theirassociation sterically or electrostatically favorable, respectively.Heterodimerization occurs between two non-identical polypeptides, e.g.two immunoglobulin heavy chains wherein the variable regions are not thesame. In some embodiments, the modification promoting heterodimerizationcomprises an amino acid mutation, specifically an amino acidsubstitution. In a particular embodiment, the modification promotingheterodimerization comprises a separate amino acid mutation,specifically an amino acid substitution, in each of the two polypeptidesdesired to form a dimer.

An “activating Fc receptor” is an Fc receptor that following engagementby an Fc region of an antibody elicits signaling events that stimulatethe receptor-bearing cell to perform effector functions. Activating Fcreceptors include FcγRIIIa (CD16a), FcγRI (CD64), FcγRIIa (CD32), andFcαRI (CD89).

The term “effector functions” when used in reference to antibodies referto those biological activities attributable to the Fc region of anantibody, which vary with the antibody isotype. Examples of antibodyeffector functions include: C1q binding and complement dependentcytotoxicity (CDC), Fc receptor binding, antibody-dependentcell-mediated cytotoxicity (ADCC), antibody-dependent cellularphagocytosis (ADCP), cytokine secretion, immune complex-mediated antigenuptake by antigen presenting cells, down regulation of cell surfacereceptors (e.g. B cell receptor), and B cell activation.

II. PD-1 Axis Binding Antagonists

Provided herein is a method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody. Also provided herein is a methodof enhancing immune function in an individual having cancer comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody. Forexample, a PD-1 axis binding antagonist includes a PD-1 bindingantagonist, a PD-L1 binding antagonist and a PD-L2 binding antagonist.PD-1 (programmed death 1) is also referred to in the art as “programmedcell death 1”, PDCD1, CD279 and SLEB2. PD-L1 (programmed death ligand 1)is also referred to in the art as “programmed cell death 1 ligand 1”,PDCD1LG1, CD274, B7-H, and PDL1. An exemplary human PD-L1 is shown inUniProtKB/Swiss-Prot Accession No.Q9NZQ7.1. PD-L2 (programmed deathligand 2) is also referred to in the art as “programmed cell death 1ligand 2”, PDCD1LG2, CD273, B7-DC, Btdc, and PDL2. An exemplary humanPD-L2 is shown in UniProtKB/Swiss-Prot Accession No. Q9BQ51. In someembodiments, PD-1, PD-L1, and PD-L2 are human PD-1, PD-L1 and PD-L2.

In some embodiments, the PD-1 binding antagonist is a molecule thatinhibits the binding of PD-1 to its ligand binding partners. In aspecific aspect the PD-1 ligand binding partners are PD-L1 and/or PD-L2.In another embodiment, a PD-L1 binding antagonist is a molecule thatinhibits the binding of PD-L1 to its binding partners. In a specificaspect, PD-L1 binding partners are PD-1 and/or B7-1. In anotherembodiment, the PD-L2 binding antagonist is a molecule that inhibits thebinding of PD-L2 to its binding partners. In a specific aspect, a PD-L2binding partner is PD-1. The antagonist may be an antibody, an antigenbinding fragment thereof, an immunoadhesin, a fusion protein, oroligopeptide.

In some embodiments, the PD-1 binding antagonist is an anti-PD-1antibody (e.g., a human antibody, a humanized antibody, or a chimericantibody). In some embodiments, the anti-PD-1 antibody is selected fromthe group consisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab),CT-011 (pidilizumab), MEDI-0680 (AMP-514), PDR001, REGN2810, andBGB-108. In some embodiments, the PD-1 binding antagonist is animmunoadhesin (e.g., an immunoadhesin comprising an extracellular orPD-1 binding portion of PD-L1 or PD-L2 fused to a constant region (e.g.,an Fc region of an immunoglobulin sequence). In some embodiments, thePD-1 binding antagonist is AMP-224. In some embodiments, the PD-L1binding antagonist is anti-PD-L1 antibody. In some embodiments, theanti-PD-L1 antibody is selected from the group consisting ofYW243.55.S70, MPDL3280A (atezolizumab), MDX-1105, MEDI4736 (durvalumab),and MSB0010718C (avelumab). Antibody YW243.55.S70 is an anti-PD-L1described in WO 2010/077634. MDX-1105, also known as BMS-936559, is ananti-PD-L1 antibody described in WO2007/005874. MEDI4736, is ananti-PD-L1 monoclonal antibody described in WO2011/066389 andUS2013/034559. Nivolumab, also known as MDX-1106-04, MDX-1106, ONO-4538,BMS-936558, and OPDIVO®, is an anti-PD-1 antibody described inWO2006/121168. Pembrolizumab, also known as MK-3475, Merck 3475,lambrolizumab, KEYTRUDA®, and SCH-900475, is an anti-PD-1 antibodydescribed in WO2009/114335. CT-011, also known as hBAT, hBAT-1 orpidilizumab, is an anti-PD-1 antibody described in WO2009/101611.AMP-224, also known as B7-DCIg, is a PD-L2-Fc fusion soluble receptordescribed in WO2010/027827 and WO2011/066342.

In some embodiments, the PD-1 axis binding antagonist is an anti-PD-L1antibody. In some embodiments, the anti-PD-L1 antibody is capable ofinhibiting binding between PD-L1 and PD-1 and/or between PD-L1 and B7-1.In some embodiments, the anti-PD-L1 antibody is a monoclonal antibody.In some embodiments, the anti-PD-L1 antibody is an antibody fragmentselected from the group consisting of Fab, Fab′-SH, Fv, scFv, and(Fab′)₂ fragments. In some embodiments, the anti-PD-L1 antibody is ahumanized antibody. In some embodiments, the anti-PD-L1 antibody is ahuman antibody.

Examples of anti-PD-L1 antibodies useful for the methods, uses,compositions and kits of this invention, and methods for making thereofare described in PCT patent application WO 2010/077634, WO2007/005874,WO2011/066389, and US2013/034559, which are incorporated herein byreference. The anti-PD-L1 antibodies useful in this invention, includingcompositions containing such antibodies, may be used in combination withan anti-CEA/anti-CD3 bispecific antibody to treat cancer.

Anti-PD1 Antibodies

In some embodiments, the anti-PD-1 antibody is MDX-1106. Alternativenames for “MDX-1106” include MDX-1106-04, ONO-4538, BMS-936558 ornivolumab. In some embodiments, the anti-PD-1 antibody is nivolumab (CASRegistry Number: 946414-94-4). In a still further embodiment, providedis an isolated anti-PD-1 antibody comprising a heavy chain variableregion comprising the heavy chain variable region amino acid sequencefrom SEQ ID NO:1 and/or a light chain variable region comprising thelight chain variable region amino acid sequence from SEQ ID NO:2. In astill further embodiment, provided is an isolated anti-PD-1 antibodycomprising a heavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 1) QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK,and

(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 2) EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC.

Anti-PD-L1 Antibodies

Anti-PD-L1 antibodies described in WO 2010/077634 A1 and U.S. Pat. No.8,217,149 may be used in the methods, uses, compositions and kitsdescribed herein. In some embodiments, the anti-PD-L1 antibody comprisesa heavy chain variable region sequence of SEQ ID NO:3 and/or a lightchain variable region sequence of SEQ ID NO:4. In a still furtherembodiment, provided is an isolated anti-PD-L1 antibody comprising aheavy chain and/or a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the heavy chain sequence:

(SEQ ID NO: 3) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRH WPGGFDYWGQGTLVTVSA,and

(b) the light chain sequences has at least 85%, at least 90%, at least91%, at least 92%, at least 93%, at least 94%, at least 95%, at least96%, at least 97%, at least 98%, at least 99% or 100% sequence identityto the light chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQ GTKVEIKR.

In one embodiment, the anti-PD-L1 antibody comprises a heavy chainvariable region polypeptide comprising an HVR-H1, HVR-H2 and HVR-H3sequence, wherein:

(SEQ ID NO: 5) (a) the HVR-H1 sequence is GFTFSX₁SWIH; (SEQ ID NO: 6)(b) the HVR-H2 sequence is AWIX₂PYGGSX₃YYADSVKG; (SEQ ID NO: 7)(c) the HVR-H3 sequence is RHWPGGFDY;further wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S. In onespecific aspect, X₁ is D; X₂ is S and X₃ is T.

In another aspect, the polypeptide further comprises variable regionheavy chain framework sequences juxtaposed between the HVRs according tothe formula:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the framework sequences are VHsubgroup III consensus framework. In a still further aspect, at leastone of the framework sequences is the following:

(SEQ ID NO: 8) HC-FR1 is EVQLVESGGGLVQPGGSLRLSCAAS (SEQ ID NO: 9)HC-FR2 is WVRQAPGKGLEWV (SEQ ID NO: 10)HC-FR3 is RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR (SEQ ID NO: 11)HC-FR4 is WGQGTLVTVSA.

In a still further aspect, the heavy chain polypeptide is furthercombined with a variable region light chain comprising an HVR-L1, HVR-L2and HVR-L3, wherein:

(SEQ ID NO: 12) (a) the HVR-L1 sequence is RASQX₄X₅X₆TX₇X₈A;(SEQ ID NO: 13) (b) the HVR-L2 sequence is SASX₉LX₁₀S,; (SEQ ID NO: 14)(c) the HVR-L3 sequence is QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;wherein: X₄ is D or V; X₅ is V or I; X₆ is S or N; X₇ is A or F; X₈ is Vor L; X₉ is F or T; X₁₀ is Y or A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y, For W; X₁₃ is Y, N, A, T, G, F or I; X₁₄ is H, V, P, T or I; X₁₅ is A, W,R, P or T. In a still further aspect, X₄ is D; X₅ is V; X₆ is S; X₇ isA; X₈ is V; X₉ is F; X₁₀ is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H;X₁₅ is A.

In a still further aspect, the light chain further comprises variableregion light chain framework sequences juxtaposed between the HVRsaccording to the formula:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the frameworksequences are VL kappa I consensus framework. In a still further aspect,at least one of the framework sequence is the following:

(SEQ ID NO: 15) LC-FR1 is DIQMTQSPSSLSASVGDRVTITC (SEQ ID NO: 16)LC-FR2 is WYQQKPGKAPKLLIY (SEQ ID NO: 17)LC-FR3 is GVPSRFSGSGSGTDFTLTISSLQPEDFATYYC (SEQ ID NO: 18)LC-FR4 is FGQGTKVEIKR.

In another embodiment, provided is an isolated anti-PD-L1 antibody orantigen binding fragment comprising a heavy chain and a light chainvariable region sequence, wherein:

(a) the heavy chain comprises and HVR-H1, HVR-H2 and HVR-H3, whereinfurther:

(SEQ ID NO: 5) (i) the HVR-H1 sequence is GFTFSX1SWIH; (SEQ ID NO: 6)(ii) the HVR-H2 sequence is AWIX2PYGGSX3YYADSVKG (SEQ ID NO: 7)(iii) the HVR-H3 sequence is RHWPGGFDY, and(b) the light chain comprises and HVR-L1, HVR-L2 and HVR-L3, whereinfurther:

(i) the HVR-L1 sequence is (SEQ ID NO: 12) RASQX₄X₅X₆TX₇X₈A(ii) the HVR-L2 sequence is (SEQ ID NO: 13) SASX₉LX₁₀S; and(iii) the HVR-L3 sequence is (SEQ ID NO: 14) QQX₁₁X₁₂X₁₃X₁₄PX₁₅T;wherein: X₁ is D or G; X₂ is S or L; X₃ is T or S; X₄ is D or V; X₅ is Vor I; X₆ is S or N; X₇ is A or F; X₈ is V or L; X₉ is F or T; X₁₀ is Yor A; X₁₁ is Y, G, F, or S; X₁₂ is L, Y, F or W; X₁₃ is Y, N, A, T, G, For I; X₁₄ is H, V, P, T or I; X₁₅ is A, W, R, P or T. In a specificaspect, X₁ is D; X₂ is S and X₃ is T. In another aspect, X₄ is D; X₅ isV; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀ is Y; X₁ i is Y; X₁₂ is L;X₁₃ is Y; X₁₄ is H; X₁₅ is A. In yet another aspect, X₁ is D; X₂ is Sand X₃ is T, X₄ is D; X₅ is V; X₆ is S; X₇ is A; X₈ is V; X₉ is F; X₁₀is Y; X₁₁ is Y; X₁₂ is L; X₁₃ is Y; X₁₄ is H and X₁₅ is A.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences are set forthas SEQ ID NOs:8, 9, 10 and 11. In a still further aspect, the lightchain framework sequences are derived from a Kabat kappa I, II, II or IVsubgroup sequence. In a still further aspect, the light chain frameworksequences are VL kappa I consensus framework. In a still further aspect,one or more of the light chain framework sequences are set forth as SEQID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region is IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

(a) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ IDNO:19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21),respectively, or(b) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ IDNO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT (SEQ ID NO:24),respectively.In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another aspect, the heavy chain variable region comprises one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences are set forth as SEQ IDNOs:8, 9, 10 and 11. In a still further aspect, the light chainframework sequences are derived from a Kabat kappa I, II, II or IVsubgroup sequence. In a still further aspect, the light chain frameworksequences are VL kappa I consensus framework. In a still further aspect,one or more of the light chain framework sequences are set forth as SEQID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region is IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In another further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWP GGFDYWGQGTLVTVSS,and/or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGT KVEIKR.In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%. In another aspect,the heavy chain variable region comprises one or more frameworksequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a further aspect, the heavy chain frameworksequences are derived from a Kabat subgroup I, II, or III sequence. In astill further aspect, the heavy chain framework sequence is a VHsubgroup III consensus framework. In a still further aspect, one or moreof the heavy chain framework sequences are set forth as SEQ ID NOs:8, 9,10 and WGQGTLVTVSS (SEQ ID NO:27).

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences are set forth as SEQ ID NOs:15, 16, 17 and 18.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region is IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect, the minimal effectorfunction results from production in prokaryotic cells. In a stillfurther specific aspect the minimal effector function results from an“effector-less Fc mutation” or aglycosylation. In still a furtherembodiment, the effector-less Fc mutation is an N297A or D265A/N297Asubstitution in the constant region.

In a further aspect, the heavy chain variable region comprises one ormore framework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In astill further aspect, the framework sequences are derived from humanconsensus framework sequences. In a still further aspect, the heavychain framework sequences are derived from a Kabat subgroup I, II, orIII sequence. In a still further aspect, the heavy chain frameworksequence is a VH subgroup III consensus framework. In a still furtheraspect, one or more of the heavy chain framework sequences is thefollowing:

HC-FR1 (SEQ ID NO: 29) EVQLVESGGGLVQPGGSLRLSCAASGFTFS HC-FR2(SEQ ID NO: 30) WVRQAPGKGLEWVA HC-FR3 (SEQ ID NO: 10)RFTISADTSKNTAYLQMNSLRAEDTAVYYCAR HC-FR4 (SEQ ID NO: 27) WGQGTLVTVSS.

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences is the following:

LC-FR1 (SEQ ID NO: 15) DIQMTQSPSSLSASVGDRVTITC LC-FR2 (SEQ ID NO: 16)WYQQKPGKAPKLLIY LC-FR3 (SEQ ID NO: 17) GVPSRFSGSGSGTDFTLTISSLQPEDFATYYCLC-FR4 (SEQ ID NO: 28) FGQGTKVEIK.

In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region is IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In yet another embodiment, provided is an anti-PD-L1 antibody comprisinga heavy chain and a light chain variable region sequence, wherein:

(c) the heavy chain further comprises an HVR-H1, HVR-H2 and an HVR-H3sequence having at least 85% sequence identity to GFTFSDSWIH (SEQ IDNO:19), AWISPYGGSTYYADSVKG (SEQ ID NO:20) and RHWPGGFDY (SEQ ID NO:21),respectively, and/or(d) the light chain further comprises an HVR-L1, HVR-L2 and an HVR-L3sequence having at least 85% sequence identity to RASQDVSTAVA (SEQ IDNO:22), SASFLYS (SEQ ID NO:23) and QQYLYHPAT (SEQ ID NO:24),respectively.In a specific aspect, the sequence identity is 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

In another aspect, the heavy chain variable region comprises one or moreframework sequences juxtaposed between the HVRs as:(HC-FR1)-(HVR-H1)-(HC-FR2)-(HVR-H2)-(HC-FR3)-(HVR-H3)-(HC-FR4), and thelight chain variable regions comprises one or more framework sequencesjuxtaposed between the HVRs as:(LC-FR1)-(HVR-L1)-(LC-FR2)-(HVR-L2)-(LC-FR3)-(HVR-L3)-(LC-FR4). In yetanother aspect, the framework sequences are derived from human consensusframework sequences. In a still further aspect, the heavy chainframework sequences are derived from a Kabat subgroup I, II, or IIIsequence. In a still further aspect, the heavy chain framework sequenceis a VH subgroup III consensus framework. In a still further aspect, oneor more of the heavy chain framework sequences are set forth as SEQ IDNOs:8, 9, 10 and WGQGTLVTVSSASTK (SEQ ID NO:31).

In a still further aspect, the light chain framework sequences arederived from a Kabat kappa I, II, II or IV subgroup sequence. In a stillfurther aspect, the light chain framework sequences are VL kappa Iconsensus framework. In a still further aspect, one or more of the lightchain framework sequences are set forth as SEQ ID NOs:15, 16, 17 and 18.In a still further specific aspect, the antibody further comprises ahuman or murine constant region. In a still further aspect, the humanconstant region is selected from the group consisting of IgG1, IgG2,IgG2, IgG3, IgG4. In a still further specific aspect, the human constantregion is IgG1. In a still further aspect, the murine constant region isselected from the group consisting of IgG1, IgG2A, IgG2B, IgG3. In astill further aspect, the murine constant region is IgG2A. In a stillfurther specific aspect, the antibody has reduced or minimal effectorfunction. In a still further specific aspect the minimal effectorfunction results from an “effector-less Fc mutation” or aglycosylation.In still a further embodiment, the effector-less Fc mutation is an N297Aor D265A/N297A substitution in the constant region.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 25) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWP GGFDYWGQGTLVTVSS,or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGT KVEIKR.

In some embodiments, provided is an isolated anti-PD-L1 antibodycomprising a heavy chain and a light chain variable region sequence,wherein the light chain variable region sequence has at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:4. In some embodiments,provided is an isolated anti-PD-L1 antibody comprising a heavy chain anda light chain variable region sequence, wherein the heavy chain variableregion sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the amino acid sequenceof SEQ ID NO:25. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein the light chain variable region sequence has at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:4 and theheavy chain variable region sequence has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO:25. In some embodiments, one, two,three, four or five amino acid residues at the N-terminal of the heavyand/or light chain may be deleted, substituted or modified.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 26) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTK,or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 4) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGT KVEIKR.

In some embodiments, provided is an isolated anti-PD-L1 antibodycomprising a heavy chain and a light chain variable region sequence,wherein the light chain variable region sequence has at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, at least 99% or 100% sequenceidentity to the amino acid sequence of SEQ ID NO:4. In some embodiments,provided is an isolated anti-PD-L1 antibody comprising a heavy chain anda light chain variable region sequence, wherein the heavy chain variableregion sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, at least 99% or 100% sequence identity to the amino acid sequenceof SEQ ID NO:26. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain variable regionsequence, wherein the light chain variable region sequence has at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%sequence identity to the amino acid sequence of SEQ ID NO:4 and theheavy chain variable region sequence has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, at least 99%, or 100% sequence identity to theamino acid sequence of SEQ ID NO:26. In some embodiments, one, two,three, four or five amino acid residues at the N-terminal of the heavyand/or light chain may be deleted, substituted or modified.

In a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 32) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG,and/or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECIn a still further embodiment, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, wherein:

(a) the heavy chain sequence has at least 85% sequence identity to theheavy chain sequence:

(SEQ ID NO: 56) EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK,and/or

(b) the light chain sequences has at least 85% sequence identity to thelight chain sequence:

(SEQ ID NO: 33) DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGEC.

In some embodiments, provided is an isolated anti-PD-L1 antibodycomprising a heavy chain and a light chain sequence, wherein the lightchain sequence has at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to the amino acid sequence of SEQID NO:33. In some embodiments, provided is an isolated anti-PD-L1antibody comprising a heavy chain and a light chain sequence, whereinthe heavy chain sequence has at least 85%, at least 86%, at least 87%,at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID NO:32 or 56. In some embodiments, provided is an isolatedanti-PD-L1 antibody comprising a heavy chain and a light chain sequence,wherein the light chain sequence has at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence of SEQ ID NO:33 and the heavy chain sequence has at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of SEQ ID NO:32 or 56. In someembodiments, provided is an isolated anti-PD-L1 antibody comprising aheavy chain and a light chain sequence, wherein the light chain sequencehas at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the amino acid sequence of SEQ ID NO:33 and theheavy chain sequence has at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof SEQ ID N0:32.

In some embodiments, the isolated anti-PD-L1 antibody is aglycosylated.Glycosylation of antibodies is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-aceylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used. Removal of glycosylation sites form anantibody is conveniently accomplished by altering the amino acidsequence such that one of the above-described tripeptide sequences (forN-linked glycosylation sites) is removed. The alteration may be made bysubstitution of an asparagine, serine or threonine residue within theglycosylation site another amino acid residue (e.g., glycine, alanine ora conservative substitution).

In any of the embodiments herein, the isolated anti-PD-L1 antibody canbind to a human PD-L1, for example a human PD-L1 as shown inUniProtKB/Swiss-Prot Accession No.Q9NZQ7.1, or a variant thereof.

In a still further embodiment, provided is an isolated nucleic acidencoding any of the antibodies described herein. In some embodiments,the nucleic acid further comprises a vector suitable for expression ofthe nucleic acid encoding any of the previously described anti-PDL1antibodies. In a still further specific aspect, the vector is in a hostcell suitable for expression of the nucleic acid. In a still furtherspecific aspect, the host cell is a eukaryotic cell or a prokaryoticcell. In a still further specific aspect, the eukaryotic cell is amammalian cell, such as Chinese hamster ovary (CHO) cell.

The antibody or antigen binding fragment thereof, may be made usingmethods known in the art, for example, by a process comprising culturinga host cell containing nucleic acid encoding any of the previouslydescribed anti-PDL1 antibodies or antigen-binding fragment in a formsuitable for expression, under conditions suitable to produce suchantibody or fragment, and recovering the antibody or fragment.

III. Anti-CEA/Anti-CD3 Bispecific Antibodies

Provided herein is a method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody. Also provided herein is a methodof enhancing immune function in an individual having cancer comprisingadministering to the individual an effective amount of a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody.

Provided herein are bispecific antibodies that bind to a humancarcinoembryonic antigen (CEA). Alternative names for “CEA” includeCEACAM5. The term “CEA” as used herein, refers to any native CEA fromany vertebrate source, including mammals such as primates (e.g. humans),non-human primates (e.g. cynomolgus monkeys) and rodents (e.g. mice andrats), unless otherwise indicated. The term encompasses “full-length”and unprocessed CEA as well as any form of CEA that results fromprocessing in the cell (e.g., mature protein). The term also encompassesnaturally occurring variants and isoforms of CEA, e.g., splice variantsor allelic variants. In one embodiment, CEA is human CEA. The amino acidsequence of human CEA is shown in UniProt (www.uniprot.org) accessionno. P06731, or NCBI (www.ncbi.nlm.nih.gov/) RefSeq NP_004354.2.

In some embodiments, the antigen binding domain of a bispecific antibodythat binds to CEA comprises a heavy chain variable region (V_(H)CEA)comprising the amino acid sequence:

(SEQ ID NO: 34) QVQLVQSGAEVKKPGASVKVSCKASGYTFTEFGMNWVRQAPGQGLEWMGWINTKTGEATYVEEFKGRVTFTTDTSTSTAYMELRSLRSDDTAVYYCARWDFA YYVEAMDYWGQGTTVTVSS,and/ora light chain variable region (V_(L)CEA) comprising the amino acidsequence:

(SEQ ID NO: 35) DIQMTQSPSSLSASVGDRVTITCKASAAVGTYVAWYQQKPGKAPKLLIYSASYRKRGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCHQYYTYPLFTFGQG TKLEIK.

In some embodiments, the antigen binding domain of a bispecific antibodythat binds to CEA comprises a heavy chain variable region comprising anHVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2 sequence ofWINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence of WDFAYYVEAMDY(SEQ ID NO:40) and a light chain variable region comprising an HVR-L1sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2 sequence of SASYRKR(SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT (SEQ ID NO:43).

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprisesa first antigen binding domain that binds a human CD3 polypeptide, and asecond antigen binding domain that binds CEA. In some embodiments, theanti-CEA/anti-CD3 bispecific antibody further comprises a third antigenbinding domain that binds CEA. In some embodiments, the second and thethird antigen binding domain are identical (i.e. have the same aminoacid sequences)

CD3 (cluster of differentiation 3) T-cell co-receptor is a proteincomplex and is composed of four distinct chains. In mammals, the complexcontains a CD3γ chain, a CD36 chain, and two CD3ε chains. These chainsassociate with the T-cell receptor (TCR) and the ζ-chain to generate anactivation signal in T lymphocytes. The TCR, ζ-chain, and CD3 moleculestogether form the TCR complex. The term “CD3” as used herein, refers toany native CD3 from any vertebrate source, including mammals such asprimates (e.g. humans), non-human primates (e.g. cynomolgus monkeys) androdents (e.g. mice and rats), unless otherwise indicated. The termencompasses “full-length” and unprocessed protein as well as any form ofthe protein or one or more of the CD3 chains (polypeptides) that resultfrom processing in the cell (e.g., mature polypeptides). The term alsoencompasses naturally occurring variants and isoforms of CD3, e.g.,splice variants or allelic variants. For example, descriptions of CD3γ,CD36, and CD3c chains and sequences are provided atwww.uniprot.org/uniprot/P04234, www.uniprot.org/uniprot/P07766, andwww.uniprot.org/uniprot/P09693. In one embodiment, CD3 is human CD3,particularly the epsilon subunit of human CD3 (CD3c). The amino acidsequence of human CD3ε is shown in UniProt (www.uniprot.org) accessionno. P07766 (version 144), or NCBI (www.ncbi.nlm.nih.gov/) RefSeqNP_000724.1. The amino acid sequence of cynomolgus [Macaca fascicularis]CD3ε is shown in NCBI GenBank no. BAB71849.1.

In some embodiments, the bispecific antibody binds to a human CD3epsilon (CD3c) polypeptide. In some embodiments, the bispecific antibodybinds to a human CD3 epsilon polypeptide in native T-cell receptor (TCR)complex in association with other TCR subunits. In some embodiments, thebispecific antibody binds to a human CD3 gamma (CD3γ) polypeptide. Insome embodiments, the bispecific antibody binds a human CD3 gammapolypeptide in native T-cell receptor (TCR) complex in association withother TCR subunits.

In one aspect, assays are provided for identifying anti-CD3 antibodiesthereof having biological activity. Biological activity may include, forexample, binding to a CD3 polypeptide (e.g., CD3 on the surface of a Tcell), or a peptide fragment thereof, either in vivo, in vitro, or exvivo. In the case of a multispecific (e.g., bispecific) anti-CD3antibody of the invention (e.g., a TCB antibody having an anti-CEAbinding domain and a binding domain that recognizes a CD3 polypeptide),biological activity may also include, for example, effector cellactivation (e.g., T cell (e.g., CD8+ and/or CD4+ T cell) activation),effector cell population expansion (i.e., an increase in T cell count),target cell population reduction (i.e., a decrease in the population ofcells expressing CEA on their cell surfaces), and/or target cellkilling. Antibodies having such biological activity in vivo and/or invitro are provided. In certain embodiments, an antibody of the inventionis tested for such biological activity.

In some embodiments, the antigen binding domain(s) of a bispecificantibody that binds to a CEA comprises a heavy chain variable region(V_(H)CEA) comprising the amino acid sequence of SEQ ID NO:34, and alight chain variable region (V_(L)CEA) comprising the amino acidsequence of SEQ ID NO:35. In some embodiments, the antigen bindingdomain(s) that binds to a CEA comprises a heavy chain variable regioncomprising an HVR-H1 sequence of EFGMN (SEQ ID NO:38), an HVR-H2sequence of WINTKTGEATYVEEFKG (SEQ ID NO:39), and an HVR-H3 sequence ofWDFAYYVEAMDY (SEQ ID NO:40) and comprises a light chain variable regioncomprising an HVR-L1 sequence of KASAAVGTYVA (SEQ ID NO:41), an HVR-L2sequence of SASYRKR (SEQ ID NO:42), and an HVR-L3 sequence of HQYYTYPLFT(SEQ ID NO:43).

In some embodiments, the antigen binding domain of a bispecific antibodythat binds to a CD3 comprises a heavy chain variable region (V_(H)CD3)comprising the amino acid sequence of SEQ ID NO:50, and a light chainvariable region (V_(L)CD3) comprising the amino acid sequence of SEQ IDNO:51. In some embodiments, the antigen binding domain that binds to aCD3 comprises a heavy chain variable region comprising an HVR-H1sequence of TYAMN (SEQ ID NO:44), an HVR-H2 sequence ofRIRSKYNNYATYYADSVKG (SEQ ID NO:45), and an HVR-H3 sequence ofHGNFGNSYVSWFAY (SEQ ID NO:46) and comprises a light chain comprising anHVR-L1 sequence of GSSTGAVTTSNYAN (SEQ ID NO:47), an HVR-L2 sequence ofGTNKRAP (SEQ ID NO:48), and an HVR-L3 sequence of ALWYSNLWV (SEQ IDNO:49).

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprisesa first antigen binding domain that binds to CD3 and a second andoptionally a third antigen binding domain that binds to CEA, wherein theantigen binding domains are Fab molecules (e.g. conventional orcrossover Fab molecules). In particular such embodiments, the bispecificantibody comprises a first antigen binding domain that binds to CD3 anda second and optionally a third antigen binding domain that binds toCEA, wherein the first antigen binding domain is a crossover Fabmolecule wherein the variable domains or the constant domains of the Fabheavy and light chain are exchanged (i.e. replaced by each other) andthe second (and third, if present) antigen binding domain is aconventional Fab molecule. In one embodiment, the first antigen bindingdomain is a crossover Fab molecule wherein the constant domains of theFab heavy and light chain are exchanged. In one embodiment, theanti-CEA/anti-CD3 bispecific antibody comprises not more than oneantigen binding domain that binds to CD3, i.e. provides monovalentbinding to CD3. In one embodiment, the first and the second antigenbinding domain are fused to each other, optionally through a peptidelinker. In some embodiments, the anti-CEA/anti-CD3 bispecific antibodycomprises a first antigen binding domain that binds to CD3 and a secondand optionally a third antigen binding domain that binds to CEA, whereinthe antigen binding domains are Fab molecules and (i) the second antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the first antigen binding domain,or (ii) the first antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the Fab heavy chain of thesecond antigen binding domain. In a particular embodiment, the secondantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingdomain. Optionally, the Fab light chain of the first antigen bindingdomain and the Fab light chain of the second antigen binding domain mayadditionally be fused to each other.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody furthercomprises an Fc domain composed of a first and a second subunit capableof stable association. In some embodiments, the anti-CEA/anti-CD3bispecific antibody comprises a first antigen binding domain that bindsto CD3, a second antigen binding domain that binds to CEA, and an Fcdomain composed of a first and a second subunit capable of stableassociation, wherein the antigen binding domains are Fab molecules andwherein (i) the second antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain, and the first antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of thefirst or second subunit of the Fc domain, or (ii) the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the Fab heavy chain of the second antigen binding domain,and the second antigen binding domain is fused at the C-terminus of theFab heavy chain to the N-terminus of the first or second subunit of theFc domain. In a particular embodiment, the second antigen binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen binding domain, and the firstantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first or second subunit of the Fc domain. Insome embodiments, the anti-CEA/anti-CD3 bispecific antibody essentiallyconsists of a first antigen binding domain that binds to CD3, a secondantigen binding domain that binds to CEA, an Fc domain composed of afirst and a second subunit capable of stable association, and optionallyone or more peptide linkers, wherein the antigen binding domains are Fabmolecules and wherein (i) the second antigen binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen binding domain, and the first antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first or second subunit of the Fc domain, or (ii) thefirst antigen binding domain is fused at the C-terminus of the Fab heavychain to the N-terminus of the Fab heavy chain of the second antigenbinding domain, and the second antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the first orsecond subunit of the Fc domain.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprisesa first antigen binding domain that binds to CD3, a second and a thirdantigen binding domain that binds to CEA, and an Fc domain composed of afirst and a second subunit capable of stable association, wherein theantigen binding domains are Fab molecules and wherein (i) the secondantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the Fab heavy chain of the first antigen bindingdomain, the first antigen binding domain is fused at the C-terminus ofthe Fab heavy chain to the N-terminus of the first subunit of the Fcdomain, and the third antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the second subunit of the Fcdomain, or (ii) the first antigen binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the second antigen binding domain, the second antigen bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and the third antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain. In a particularembodiment, the second antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain, the first antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and the third antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain. In some embodiments, the anti-CEA/anti-CD3bispecific antibody essentially consists of a first antigen bindingdomain that binds to CD3, a second and a third antigen binding domainthat binds to CEA, an Fc domain composed of a first and a second subunitcapable of stable association, and optionally one or more peptidelinkers, wherein the antigen binding domains are Fab molecules andwherein (i) the second antigen binding domain is fused at the C-terminusof the Fab heavy chain to the N-terminus of the Fab heavy chain of thefirst antigen binding domain, the first antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and the third antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the secondsubunit of the Fc domain, or (ii) the first antigen binding domain isfused at the C-terminus of the Fab heavy chain to the N-terminus of theFab heavy chain of the second antigen binding domain, the second antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and the third antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the second subunit of the Fc domain.

In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises

(i) a first antigen binding domain that binds to CD3, comprising a heavychain variable region (V_(H)CD3) comprising HVR-H1 sequence of SEQ IDNO:44, HVR-H2 sequence of SEQ ID NO:45, and HVR-H3 sequence of SEQ IDNO:46; and a light chain variable region (V_(L)CD3) comprising HVR-L1sequence of SEQ ID NO:47, HVR-L2 sequence of SEQ ID NO:48, and HVR-L3sequence of SEQ ID NO:49, wherein the first antigen binding moiety is acrossover Fab molecule wherein either the variable or the constantregions, particularly the constant regions, of the Fab light chain andthe Fab heavy chain are exchanged;(ii) a second and a third antigen binding domain that bind to CEA,comprising a heavy chain variable region (V_(H)CEA) comprising HVR-H1sequence of SEQ ID NO:38, HVR-H2 sequence of SEQ ID NO:39, and HVR-H3sequence of SEQ ID NO:40; and a light chain variable region (V_(L)CEA)comprising HVR-L1 sequence of SEQ ID NO:41, HVR-L2 sequence of SEQ IDNO:42, and HVR-L3 sequence of SEQ ID NO:43, wherein the second and thirdantigen binding moiety are each a Fab molecule, particularly aconventional Fab molecule;(iii) an Fc domain composed of a first and a second subunit capable ofstable association, wherein the second antigen binding domain is fusedat the C-terminus of the Fab heavy chain to the N-terminus of the Fabheavy chain of the first antigen binding domain, and the first antigenbinding domain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdantigen binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.

The Fab molecules may be fused to the Fc domain or to each otherdirectly or through a peptide linker, comprising one or more aminoacids, typically about 2-20 amino acids. Peptide linkers are known inthe art and are described herein. Suitable, non-immunogenic peptidelinkers include, for example, (G₄S)_(n), (SG₄)_(n), (G₄S)_(n) orG₄(SG₄)_(n) peptide linkers. “n” is generally an integer from 1 to 10,typically from 2 to 4. In one embodiment said peptide linker has alength of at least 5 amino acids, in one embodiment a length of 5 to100, in a further embodiment of 10 to 50 amino acids. In one embodimentsaid peptide linker is (GxS)_(n) or (GxS)_(n)G_(m) with G=glycine,S=serine, and (x=3, n=3, 4, 5 or 6, and m=0, 1, 2 or 3) or (x=4, n=2, 3,4 or 5 and m=0, 1, 2 or 3), in one embodiment x=4 and n=2 or 3, in afurther embodiment x=4 and n=2. In one embodiment said peptide linker is(G45)₂. A particularly suitable peptide linker for fusing the Fab lightchains of the first and the second antigen binding domain to each otheris (G45)₂. An exemplary peptide linker suitable for connecting the Fabheavy chains of the first and the second antigen binding domaincomprises the sequence (D)-(G₄S)₂. Another suitable such linkercomprises the sequence (G₄S)₄. Additionally, linkers may comprise (aportion of) an immunoglobulin hinge region. Particularly where a Fabmolecule is fused to the N-terminus of an Fc domain subunit, it may befused via an immunoglobulin hinge region or a portion thereof, with orwithout an additional peptide linker.

In some embodiments, the anti-CEA/anti-CD3 bispecific antibody comprises(i) a polypeptide wherein the Fab heavy chain of a first Fab moleculeshares a carboxy-terminal peptide bond with the Fab heavy chain variableregion of a second Fab molecule which in turn shares a carboxy-terminalpeptide bond with the Fab light chain constant region of the second Fabmolecule (i.e. the second Fab molecule comprises a crossover Fab heavychain, wherein the heavy chain constant region is replaced by a lightchain constant region), which in turn shares a carboxy-terminal peptidebond with a first Fc domain subunit(VH₍₁₎-CH1₍₁₎-VH₍₂₎-CL₍₂₎-CH2-CH3(-CH4)), (ii) a crossover Fab lightchain polypeptide of the second Fab molecule, wherein the Fab lightchain variable region of the second Fab molecule shares acarboxy-terminal peptide bond with the Fab heavy chain constant regionof the second Fab molecule (VL₍₂₎-CH1₍₂₎), (iii) the Fab light chainpolypeptide of the first Fab molecule (VL₍₁₎-CL₍₁₎), (iv) a polypeptidewherein the Fab heavy chain of a third Fab molecule shares acarboxy-terminal peptide bond with a second Fc domain subunit(VH₍₃₎-CH1₍₃₎-CH2-CH3(-CH4)) and (v) the Fab light chain polypeptide ofthe third Fab molecule (VL₍₃₎-CL₍₃₎). In certain embodiments, the Fablight chain polypeptide of the first and the third Fab molecule areidentical. In certain embodiments the polypeptides are covalentlylinked, e.g., by a disulfide bond. In certain embodiments, the first andthe third Fab molecule bind to CEA, and the second Fab molecule binds toCD3.

In one embodiment the anti-CEA/anti-CD3 bispecific antibody comprises apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 52, apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 53, apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 54, and apolypeptide comprising a sequence that is at least 80%, 85%, 90%, 95%,96%, 97%, 98%, or 99% identical to the sequence of SEQ ID NO: 55. In oneembodiment, the bispecific antibody comprises a polypeptide comprisingthe sequence of SEQ ID NO: 52, a polypeptide comprising the sequence ofSEQ ID NO: 53, a polypeptide comprising the sequence of SEQ ID NO: 54,and a polypeptide comprising the sequence of SEQ ID NO: 55.

Fc Domain

The anti-CEA/anti-CD3 bispecific antibody used in the present inventionmay comprise an Fc domain which consists of a pair of polypeptide chainscomprising heavy chain domains of an antibody molecule. For example, theFc domain of an immunoglobulin G (IgG) molecule is a dimer, each subunitof which comprises the CH2 and CH3 IgG heavy chain constant domains. Thetwo subunits of the Fc domain are capable of stable association witheach other.

In one embodiment, the Fc domain is an IgG Fc domain. In a particularembodiment the Fc domain is an IgG₁ Fc domain. In another embodiment theFc domain is an IgG₄ Fc domain. In a more specific embodiment, the Fcdomain is an IgG₄ Fc domain comprising an amino acid substitution atposition S228 (EU numbering), particularly the amino acid substitutionS228P. This amino acid substitution reduces in vivo Fab arm exchange ofIgG4 antibodies (see Stubenrauch et al., Drug Metabolism and Disposition38, 84-91 (2010)). In a further particular embodiment the Fc domain ishuman. An exemplary sequence of a human IgG₁ Fc region is given in SEQID NO: 57.

Fc Domain Modifications Promoting Heterodimerization

The anti-CEA/anti-CD3 bispecific antibody used in the present inventionmay comprise different components (e.g. antigen binding domains) fusedto one or the other of the two subunits of the Fc domain, thus the twosubunits of the Fc domain are typically comprised in two non-identicalpolypeptide chains. Recombinant co-expression of these polypeptides andsubsequent dimerization leads to several possible combinations of thetwo polypeptides. To improve the yield and purity of such antibodies inrecombinant production, it will thus be advantageous to introduce in theFc domain of the antibody a modification promoting the association ofthe desired polypeptides.

Accordingly, in particular embodiments the Fc domain comprises amodification promoting the association of the first and the secondsubunit of the Fc domain. The site of most extensive protein-proteininteraction between the two subunits of a human IgG Fc domain is in theCH3 domain of the Fc domain. Thus, in one embodiment said modificationis in the CH3 domain of the Fc domain.

There exist several approaches for modifications in the CH3 domain ofthe Fc domain in order to enforce heterodimerization, which are welldescribed e.g. in WO 96/27011, WO 98/050431, EP 1870459, WO 2007/110205,WO 2007/147901, WO 2009/089004, WO 2010/129304, WO 2011/90754, WO2011/143545, WO 2012058768, WO 2013157954, WO 2013096291. Typically, inall such approaches the CH3 domain of the first subunit of the Fc domainand the CH3 domain of the second subunit of the Fc domain are bothengineered in a complementary manner so that each CH3 domain (or theheavy chain comprising it) can no longer homodimerize with itself but isforced to heterodimerize with the complementarily engineered other CH3domain (so that the first and second CH3 domain heterodimerize and nohomodimers between the two first or the two second CH3 domains areformed).

In a specific embodiment said modification promoting the association ofthe first and the second subunit of the Fc domain is a so-called“knob-into-hole” modification, comprising a “knob” modification in oneof the two subunits of the Fc domain and a “hole” modification in theother one of the two subunits of the Fc domain.

The knob-into-hole technology is described e.g. in U.S. Pat. Nos.5,731,168; 7,695,936; Ridgway et al., Prot Eng 9, 617-621 (1996) andCarter, J Immunol Meth 248, 7-15 (2001). Generally, the method involvesintroducing a protuberance (“knob”) at the interface of a firstpolypeptide and a corresponding cavity (“hole”) in the interface of asecond polypeptide, such that the protuberance can be positioned in thecavity so as to promote heterodimer formation and hinder homodimerformation. Protuberances are constructed by replacing small amino acidside chains from the interface of the first polypeptide with larger sidechains (e.g. tyrosine or tryptophan). Compensatory cavities of identicalor similar size to the protuberances are created in the interface of thesecond polypeptide by replacing large amino acid side chains withsmaller ones (e.g. alanine or threonine).

Accordingly, in a particular embodiment, in the CH3 domain of the firstsubunit of the Fc domain an amino acid residue is replaced with an aminoacid residue having a larger side chain volume, thereby generating aprotuberance within the CH3 domain of the first subunit which ispositionable in a cavity within the CH3 domain of the second subunit,and in the CH3 domain of the second subunit of the Fc domain an aminoacid residue is replaced with an amino acid residue having a smallerside chain volume, thereby generating a cavity within the CH3 domain ofthe second subunit within which the protuberance within the CH3 domainof the first subunit is positionable.

Preferably said amino acid residue having a larger side chain volume isselected from the group consisting of arginine (R), phenylalanine (F),tyrosine (Y), and tryptophan (W).

Preferably said amino acid residue having a smaller side chain volume isselected from the group consisting of alanine (A), serine (S), threonine(T), and valine (V).

The protuberance and cavity can be made by altering the nucleic acidencoding the polypeptides, e.g. by site-specific mutagenesis, or bypeptide synthesis.

In a specific embodiment, in the CH3 domain of the first subunit of theFc domain (the “knobs” subunit) the threonine residue at position 366 isreplaced with a tryptophan residue (T366W), and in the CH3 domain of thesecond subunit of the Fc domain (the “hole” subunit) the tyrosineresidue at position 407 is replaced with a valine residue (Y407V). Inone embodiment, in the second subunit of the Fc domain additionally thethreonine residue at position 366 is replaced with a serine residue(T366S) and the leucine residue at position 368 is replaced with analanine residue (L368A) (EU numbering).

In yet a further embodiment, in the first subunit of the Fc domainadditionally the serine residue at position 354 is replaced with acysteine residue (S354C) or the glutamic acid residue at position 356 isreplaced with a cysteine residue (E356C), and in the second subunit ofthe Fc domain additionally the tyrosine residue at position 349 isreplaced by a cysteine residue (Y349C) (EU numbering). Introduction ofthese two cysteine residues results in formation of a disulfide bridgebetween the two subunits of the Fc domain, further stabilizing the dimer(Carter, J Immunol Methods 248, 7-15 (2001)).

In a particular embodiment, the first subunit of the Fc domain comprisesamino acid substitutions S354C and T366W, and the second subunit of theFc domain comprises amino acid substitutions Y349C, T366S, L368A andY407V (EU numbering).

In a particular embodiment the antigen binding domain that binds to CD3in the anti-CEA/anti-CD3 bispecific antibody described herein is fusedto the first subunit of the Fc domain (comprising the “knob”modification). Without wishing to be bound by theory, fusion of the CD3binding domain to the knob-containing subunit of the Fc domain will(further) minimize the generation of bispecific antibodies comprisingtwo CD3 binding domains (steric clash of two knob-containingpolypeptides).

Other techniques of CH3-modification for enforcing theheterodimerization are contemplated as alternatives according to theinvention and are described e.g. in WO 96/27011, WO 98/050431, EP1870459, WO 2007/110205, WO 2007/147901, WO 2009/089004, WO 2010/129304,WO 2011/90754, WO 2011/143545, WO 2012/058768, WO 2013/157954, WO2013/096291.

In one embodiment the heterodimerization approach described in EP1870459 A1, is used alternatively. This approach is based on theintroduction of charged amino acids with opposite charges at specificamino acid positions in the CH3/CH3 domain interface between the twosubunits of the Fc domain. One preferred embodiment are amino acidmutations R409D; K370E in one of the two CH3 domains (of the Fc domain)and amino acid mutations D399K; E357K in the other one of the CH3domains of the Fc domain (EU numbering).

In another embodiment the antibody comprises amino acid mutation T366Win the CH3 domain of the first subunit of the Fc domain and amino acidmutations T366S, L368A, Y407V in the CH3 domain of the second subunit ofthe Fc domain, and additionally amino acid mutations R409D; K370E in theCH3 domain of the first subunit of the Fc domain and amino acidmutations D399K; E357K in the CH3 domain of the second subunit of the Fcdomain (EU numbering).

In another embodiment the antibody comprises amino acid mutations S354C,T366W in the CH3 domain of the first subunit of the Fc domain and aminoacid mutations Y349C, T366S, L368A, Y407V in the CH3 domain of thesecond subunit of the Fc domain, or the antibody comprises amino acidmutations Y349C, T366W in the CH3 domain of the first subunit of the Fcdomain and amino acid mutations S354C, T366S, L368A, Y407V in the CH3domains of the second subunit of the Fc domain and additionally aminoacid mutations R409D; K370E in the CH3 domain of the first subunit ofthe Fc domain and amino acid mutations D399K; E357K in the CH3 domain ofthe second subunit of the Fc domain (all numberings according to EUnumbering).

In one embodiment the heterodimerization approach described in WO2013/157953 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366K and a second CH3 domain comprisesamino acid mutation L351D (EU numbering). In a further embodiment thefirst CH3 domain comprises further amino acid mutation L351K. In afurther embodiment the second CH3 domain comprises further an amino acidmutation selected from Y349E, Y349D and L368E (preferably L368E) (EUnumbering).

In one embodiment the heterodimerization approach described in WO2012/058768 is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutations L351Y, Y407A and a second CH3 domaincomprises amino acid mutations T366A, K409F. In a further embodiment thesecond CH3 domain comprises a further amino acid mutation at positionT411, D399, 5400, F405, N390, or K392, e.g. selected from a) T411N,T411R, T411Q, T411K, T411D, T411E or T411W, b) D399R, D399W, D399Y orD399K, c) S400E, S400D, S400R, or S400K, d) F4051, F405M, F405T, F405S,F405V or F405W, e) N390R, N390K or N390D, f) K392V, K392M, K392R, K392L,K392F or K392E (EU numbering). In a further embodiment a first CH3domain comprises amino acid mutations L351Y, Y407A and a second CH3domain comprises amino acid mutations T366V, K409F. In a furtherembodiment a first CH3 domain comprises amino acid mutation Y407A and asecond CH3 domain comprises amino acid mutations T366A, K409F. In afurther embodiment the second CH3 domain further comprises amino acidmutations K392E, T411E, D399R and S400R (EU numbering).

In one embodiment the heterodimerization approach described in WO2011/143545 is used alternatively, e.g. with the amino acid modificationat a position selected from the group consisting of 368 and 409 (EUnumbering).

In one embodiment the heterodimerization approach described in WO2011/090762, which also uses the knobs-into-holes technology describedabove, is used alternatively. In one embodiment a first CH3 domaincomprises amino acid mutation T366W and a second CH3 domain comprisesamino acid mutation Y407A. In one embodiment a first CH3 domaincomprises amino acid mutation T366Y and a second CH3 domain comprisesamino acid mutation Y407T (EU numbering).

In one embodiment the antibody or its Fc domain is of IgG2 subclass andthe heterodimerization approach described in WO 2010/129304 is usedalternatively.

In an alternative embodiment a modification promoting association of thefirst and the second subunit of the Fc domain comprises a modificationmediating electrostatic steering effects, e.g. as described in PCTpublication WO 2009/089004. Generally, this method involves replacementof one or more amino acid residues at the interface of the two Fc domainsubunits by charged amino acid residues so that homodimer formationbecomes electrostatically unfavorable but heterodimerizationelectrostatically favorable. In one such embodiment a first CH3 domaincomprises amino acid substitution of K392 or N392 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D),preferably K392D or N392D) and a second CH3 domain comprises amino acidsubstitution of D399, E356, D356, or E357 with a positively chargedamino acid (e.g. lysine (K) or arginine (R), preferably D399K, E356K,D356K, or E357K, and more preferably D399K and E356K). In a furtherembodiment the first CH3 domain further comprises amino acidsubstitution of K409 or R409 with a negatively charged amino acid (e.g.glutamic acid (E), or aspartic acid (D), preferably K409D or R409D). Ina further embodiment the first CH3 domain further or alternativelycomprises amino acid substitution of K439 and/or K370 with a negativelycharged amino acid (e.g. glutamic acid (E), or aspartic acid (D)) (allnumberings according to EU numbering).

In yet a further embodiment the heterodimerization approach described inWO 2007/147901 is used alternatively. In one embodiment a first CH3domain comprises amino acid mutations K253E, D282K, and K322D and asecond CH3 domain comprises amino acid mutations D239K, E240K, and K292D(EU numbering).

In still another embodiment the heterodimerization approach described inWO 2007/110205 can be used alternatively.

In one embodiment, the first subunit of the Fc domain comprises aminoacid substitutions K392D and K409D, and the second subunit of the Fcdomain comprises amino acid substitutions D356K and D399K (EUnumbering).

Fc Domain Modifications Reducing Fc Receptor Binding and/or EffectorFunction

The Fc domain confers to an antibody, such as a bispecific antibody,favorable pharmacokinetic properties, including a long serum half-lifewhich contributes to good accumulation in the target tissue and afavorable tissue-blood distribution ratio. At the same time it may,however, lead to undesirable targeting of the antibody to cellsexpressing Fc receptors rather than to the preferred antigen-bearingcells. Moreover, the co-activation of Fc receptor signaling pathways maylead to cytokine release which, in combination with otherimmunostimulatory properties the antibody may have and the longhalf-life of the antibody, results in excessive activation of cytokinereceptors and severe side effects upon systemic administration.

Accordingly, in particular embodiments, the Fc domain of theanti-CEA/anti-CD3 bispecific antibody exhibits reduced binding affinityto an Fc receptor and/or reduced effector function, as compared to anative IgG₁ Fc domain. In one such embodiment the Fc domain (or themolecule, e.g. antibody, comprising said Fc domain) exhibits less than50%, preferably less than 20%, more preferably less than 10% and mostpreferably less than 5% of the binding affinity to an Fc receptor, ascompared to a native IgG₁ Fc domain (or a corresponding moleculecomprising a native IgG₁ Fc domain), and/or less than 50%, preferablyless than 20%, more preferably less than 10% and most preferably lessthan 5% of the effector function, as compared to a native IgG₁ Fc domaindomain (or a corresponding molecule comprising a native IgG₁ Fc domain).In one embodiment, the Fc domain (or the molecule, e.g. antibody,comprising said Fc domain) does not substantially bind to an Fc receptorand/or induce effector function. In a particular embodiment the Fcreceptor is an Fey receptor. In one embodiment the Fc receptor is ahuman Fc receptor. In one embodiment the Fc receptor is an activating Fcreceptor. In a specific embodiment the Fc receptor is an activatinghuman Fcγ receptor, more specifically human FcγRIIIa, FcγRI or FcγRIIa,most specifically human FcγRIIIa. In one embodiment the effectorfunction is one or more selected from the group of CDC, ADCC, ADCP, andcytokine secretion. In a particular embodiment the effector function isADCC. In one embodiment the Fc domain exhibits substantially similarbinding affinity to neonatal Fc receptor (FcRn), as compared to a nativeIgG₁ Fc domain domain. Substantially similar binding to FcRn is achievedwhen the Fc domain (or the molecule, e.g. antibody, comprising said Fcdomain) exhibits greater than about 70%, particularly greater than about80%, more particularly greater than about 90% of the binding affinity ofa native IgG₁ Fc domain (or the corresponding molecule comprising anative IgG₁ Fc domain) to FcRn.

In certain embodiments the Fc domain is engineered to have reducedbinding affinity to an Fc receptor and/or reduced effector function, ascompared to a non-engineered Fc domain. In particular embodiments, theFc domain comprises one or more amino acid mutation that reduces thebinding affinity of the Fc domain to an Fc receptor and/or effectorfunction. Typically, the same one or more amino acid mutation is presentin each of the two subunits of the Fc domain. In one embodiment theamino acid mutation reduces the binding affinity of the Fc domain to anFc receptor. In one embodiment the amino acid mutation reduces thebinding affinity of the Fc domain to an Fc receptor by at least 2-fold,at least 5-fold, or at least 10-fold. In embodiments where there is morethan one amino acid mutation that reduces the binding affinity of the Fcdomain to the Fc receptor, the combination of these amino acid mutationsmay reduce the binding affinity of the Fc domain to an Fc receptor by atleast 10-fold, at least 20-fold, or even at least 50-fold. In oneembodiment the molecule, e.g. antibody, comprising an engineered Fcdomain exhibits less than 20%, particularly less than 10%, moreparticularly less than 5% of the binding affinity to an Fc receptor ascompared to a corresponding molecule comprising a non-engineered Fcdomain. In a particular embodiment the Fc receptor is an Fey receptor.In some embodiments the Fc receptor is a human Fc receptor. In someembodiments the Fc receptor is an activating Fc receptor. In a specificembodiment the Fc receptor is an activating human Fcγ receptor, morespecifically human FcγRIIIa, FcγRI or FcγRIIa, most specifically humanFcγRIIIa. Preferably, binding to each of these receptors is reduced. Insome embodiments binding affinity to a complement component,specifically binding affinity to C1q, is also reduced. In one embodimentbinding affinity to neonatal Fc receptor (FcRn) is not reduced.Substantially similar binding to FcRn, i.e. preservation of the bindingaffinity of the Fc domain to said receptor, is achieved when the Fcdomain (or the molecule, e.g. antibody, comprising said Fc domain)exhibits greater than about 70% of the binding affinity of anon-engineered form of the Fc domain (or a corresponding moleculecomprising said non-engineered form of the Fc domain) to FcRn. The Fcdomain, or molecule (e.g. antibody) comprising said Fc domain, mayexhibit greater than about 80% and even greater than about 90% of suchaffinity. In certain embodiments the Fc domain is engineered to havereduced effector function, as compared to a non-engineered Fc domain.The reduced effector function can include, but is not limited to, one ormore of the following: reduced complement dependent cytotoxicity (CDC),reduced antibody-dependent cell-mediated cytotoxicity (ADCC), reducedantibody-dependent cellular phagocytosis (ADCP), reduced cytokinesecretion, reduced immune complex-mediated antigen uptake byantigen-presenting cells, reduced binding to NK cells, reduced bindingto macrophages, reduced binding to monocytes, reduced binding topolymorphonuclear cells, reduced direct signaling inducing apoptosis,reduced crosslinking of target-bound antibodies, reduced dendritic cellmaturation, or reduced T cell priming. In one embodiment the reducedeffector function is one or more selected from the group of reduced CDC,reduced ADCC, reduced ADCP, and reduced cytokine secretion. In aparticular embodiment the reduced effector function is reduced ADCC. Inone embodiment the reduced ADCC is less than 20% of the ADCC induced bya non-engineered Fc domain (or a corresponding molecule comprising anon-engineered Fc domain).

In one embodiment the amino acid mutation that reduces the bindingaffinity of the Fc domain to an Fc receptor and/or effector function isan amino acid substitution. In one embodiment the Fc domain comprises anamino acid substitution at a position selected from the group of E233,L234, L235, N297, P331 and P329 (EU numbering). In a more specificembodiment the Fc domain comprises an amino acid substitution at aposition selected from the group of L234, L235 and P329 (EU numbering).In some embodiments the Fc domain comprises the amino acid substitutionsL234A and L235A (EU numbering). In one such embodiment, the Fc domain isan IgG₁ Fc domain, particularly a human IgG₁ Fc domain. In oneembodiment the Fc domain comprises an amino acid substitution atposition P329. In a more specific embodiment the amino acid substitutionis P329A or P329G, particularly P329G (EU numbering). In one embodimentthe Fc domain comprises an amino acid substitution at position P329 anda further amino acid substitution at a position selected from E233,L234, L235, N297 and P331 (EU numbering). In a more specific embodimentthe further amino acid substitution is E233P, L234A, L235A, L235E,N297A, N297D or P331S. In particular embodiments the Fc domain comprisesamino acid substitutions at positions P329, L234 and L235 EU numbering).In more particular embodiments the Fc domain comprises the amino acidmutations L234A, L235A and P329G (“P329G LALA”). In one such embodiment,the Fc domain is an IgG₁ Fc domain, particularly a human IgG₁ Fc domain.The “P329G LALA” combination of amino acid substitutions almostcompletely abolishes Fcγ receptor (as well as complement) binding of ahuman IgG₁ Fc domain, as described in PCT publication no. WO2012/130831, incorporated herein by reference in its entirety. WO2012/130831 also describes methods of preparing such mutant Fc domainsand methods for determining its properties such as Fc receptor bindingor effector functions.

IgG₄ antibodies exhibit reduced binding affinity to Fc receptors andreduced effector functions as compared to IgG₁ antibodies. Hence, insome embodiments the Fc domain is an IgG₄ Fc domain, particularly ahuman IgG₄ Fc domain. In one embodiment the IgG₄ Fc domain comprisesamino acid substitutions at position 5228, specifically the amino acidsubstitution S228P (EU numbering). To further reduce its bindingaffinity to an Fc receptor and/or its effector function, in oneembodiment the IgG₄ Fc domain comprises an amino acid substitution atposition L235, specifically the amino acid substitution L235E (EUnumbering). In another embodiment, the IgG₄ Fc domain comprises an aminoacid substitution at position P329, specifically the amino acidsubstitution P329G (EU numbering). In a particular embodiment, the IgG₄Fc domain comprises amino acid substitutions at positions S228, L235 andP329, specifically amino acid substitutions S228P, L235E and P329G (EUnumbering). Such IgG₄ Fc domain mutants and their Fcγ receptor bindingproperties are described in PCT publication no. WO 2012/130831,incorporated herein by reference in its entirety.

In a particular embodiment the Fc domain exhibiting reduced bindingaffinity to an Fc receptor and/or reduced effector function, as comparedto a native IgG₁ Fc domain, is a human IgG₁ Fc domain comprising theamino acid substitutions L234A, L235A and optionally P329G, or a humanIgG₄ Fc domain comprising the amino acid substitutions S228P, L235E andoptionally P329G (EU numbering).

In certain embodiments N-glycosylation of the Fc domain has beeneliminated. In one such embodiment the Fc domain comprises an amino acidmutation at position N297, particularly an amino acid substitutionreplacing asparagine by alanine (N297A) or aspartic acid (N297D) orglycine (N297G) (EU numbering).

In addition to the Fc domains described hereinabove and in PCTpublication no. WO 2012/130831, Fc domains with reduced Fc receptorbinding and/or effector function also include those with substitution ofone or more of Fc domain residues 238, 265, 269, 270, 297, 327 and 329(U.S. Pat. No. 6,737,056) (EU numbering). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Mutant Fc domains can be prepared by amino acid deletion, substitution,insertion or modification using genetic or chemical methods well knownin the art. Genetic methods may include site-specific mutagenesis of theencoding DNA sequence, PCR, gene synthesis, and the like. The correctnucleotide changes can be verified for example by sequencing.

Binding to Fc receptors can be easily determined e.g. by ELISA, or bySurface Plasmon Resonance (SPR) using standard instrumentation such as aBIAcore instrument (GE Healthcare), and Fc receptors such as may beobtained by recombinant expression. Alternatively, binding affinity ofFc domains or molecules comprising an Fc domain for Fc receptors may beevaluated using cell lines known to express particular Fc receptors,such as human NK cells expressing FcγIIIa receptor.

Effector function of an Fc domain, or a molecule (e.g. an antibody)comprising an Fc domain, can be measured by methods known in the art. Asuitable assay for measuring ADCC is described herein. Other examples ofin vitro assays to assess ADCC activity of a molecule of interest aredescribed in U.S. Pat. No. 5,500,362; Hellstrom et al. Proc Natl AcadSci USA 83, 7059-7063 (1986) and Hellstrom et al., Proc Natl Acad SciUSA 82, 1499-1502 (1985); U.S. Pat. No. 5,821,337; Bruggemann et al., JExp Med 166, 1351-1361 (1987). Alternatively, non-radioactive assaysmethods may be employed (see, for example, ACTI™ non-radioactivecytotoxicity assay for flow cytometry (CellTechnology, Inc. MountainView, Calif.); and CytoTox 96® non-radioactive cytotoxicity assay(Promega, Madison, Wis.)). Useful effector cells for such assays includeperipheral blood mononuclear cells (PBMC) and Natural Killer (NK) cells.Alternatively, or additionally, ADCC activity of the molecule ofinterest may be assessed in vivo, e.g. in a animal model such as thatdisclosed in Clynes et al., Proc Natl Acad Sci USA 95, 652-656 (1998).

In some embodiments, binding of the Fc domain to a complement component,specifically to C1q, is reduced. Accordingly, in some embodimentswherein the Fc domain is engineered to have reduced effector function,said reduced effector function includes reduced CDC. C1q binding assaysmay be carried out to determine whether the Fc domain, or molecule (e.g.antibody) comprising the Fc domain, is able to bind C1q and hence hasCDC activity. See e.g., C1q and C3c binding ELISA in WO 2006/029879 andWO 2005/100402. To assess complement activation, a CDC assay may beperformed (see, for example, Gazzano-Santoro et al., J Immunol Methods202, 163 (1996); Cragg et al., Blood 101, 1045-1052 (2003); and Craggand Glennie, Blood 103, 2738-2743 (2004)).

In some embodiments, the bispecific antibody is a single-chainbispecific antibody comprising the first antigen binding domain and thesecond antigen binding domain. In some embodiments, the single-chainbispecific antibody comprises variable regions, as arranged fromN-terminus to C-terminus, selected from the group consisting of (1)V_(H)CEA-V_(L)CEA-V_(H)CD3-V_(L)CD3, (2)V_(H)CD3-V_(L)CD3-V_(H)CEA-V_(L)CEA, (3)V_(H)CD3-V_(L)CD3-V_(L)CEA-V_(H)CEA, (4)V_(H)CEA-V_(L)CEA-V_(L)CD3-V_(H)CD3, (5)V_(L)CEA-V_(H)CEA-V_(H)CD3-V_(L)CD3, and (6)V_(L)CD3-V_(H)CD3-V_(H)CEA-V_(L)CEA.

IV. Antibody Preparation

Antibodies described herein are prepared using techniques available inthe art for generating antibodies, exemplary methods of which aredescribed in more detail in the following sections.

The antibody is directed against an antigen of interest (i.e., PD-L1(such as a human PD-L1), CEA, or CD3 (such as a human CD3)). Preferably,the antigen is a biologically important polypeptide and administrationof the antibody to a mammal suffering from a disorder can result in atherapeutic benefit in that mammal.

In certain embodiments, an antibody provided herein has a dissociationconstant (K_(D)) of ≤1 μM, ≤150 nM, ≤100 nM, ≤50 nM, ≤10 nM, ≤1 nM, ≤0.1nM, ≤0.01 nM, or ≤0.001 nM (e.g. 10⁻⁸M or less, e.g. from 10⁻⁸M to10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M).

In one embodiment, K_(D) is measured by a radiolabeled antigen bindingassay (RIA) performed with the Fab version of an antibody of interestand its antigen as described by the following assay. Solution bindingaffinity of Fabs for antigen is measured by equilibrating Fab with aminimal concentration of (¹²⁵I)-labeled antigen in the presence of atitration series of unlabeled antigen, then capturing bound antigen withan anti-Fab antibody-coated plate (see, e.g., Chen et al., J. Mol. Biol.293:865-881(1999)). To establish conditions for the assay, MICROTITER®multi-well plates (Thermo Scientific) are coated overnight with 5 μg/mlof a capturing anti-Fab antibody (Cappel Labs) in 50 mM sodium carbonate(pH 9.6), and subsequently blocked with 2% (w/v) bovine serum albumin inPBS for two to five hours at room temperature (approximately 23° C.). Ina non-adsorbent plate (Nunc #269620), 100 pM or 26 pM [¹²⁵I]-antigen aremixed with serial dilutions of a Fab of interest. The Fab of interest isthen incubated overnight; however, the incubation may continue for alonger period (e.g., about 65 hours) to ensure that equilibrium isreached. Thereafter, the mixtures are transferred to the capture platefor incubation at room temperature (e.g., for one hour). The solution isthen removed and the plate washed eight times with 0.1% polysorbate 20(TWEEN-20®) in PBS. When the plates have dried, 150 μl/well ofscintillant (MICROSCINT-20™; Packard) is added, and the plates arecounted on a TOPCOUNT™ gamma counter (Packard) for ten minutes.Concentrations of each Fab that give less than or equal to 20% ofmaximal binding are chosen for use in competitive binding assays.

According to another embodiment, K_(D) is measured using surface plasmonresonance assays using a BIACORE®-2000 or a BIACORE®-3000 (BIAcore,Inc., Piscataway, N.J.) at 25° C. with immobilized antigen CMS chips at˜10 response units (RU). Briefly, carboxymethylated dextran biosensorchips (CMS, BIACORE, Inc.) are activated withN-ethyl-N′-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) andN-hydroxysuccinimide (NHS) according to the supplier's instructions.Antigen is diluted with 10 mM sodium acetate, pH 4.8, to 5 μg/ml (˜0.2μM) before injection at a flow rate of 5 μl/minute to achieveapproximately 10 response units (RU) of coupled protein. Following theinjection of antigen, 1 M ethanolamine is injected to block unreactedgroups. For kinetics measurements, two-fold serial dilutions of Fab(0.78 nM to 500 nM) are injected in PBS with 0.05% polysorbate 20(TWEEN-20™) surfactant (PBST) at 25° C. at a flow rate of approximately25 μl/min. Association rates (k_(on)) and dissociation rates (k_(off))are calculated using a simple one-to-one Langmuir binding model(BIACORE® Evaluation Software version 3.2) by simultaneously fitting theassociation and dissociation sensorgrams. The equilibrium dissociationconstant (K_(D)) is calculated as the ratio k_(off)/k_(on). See, e.g.,Chen et al., J. Mol. Biol. 293:865-881 (1999). If the on-rate exceeds10⁶ M⁻¹ s⁻¹ by the surface plasmon resonance assay above, then theon-rate can be determined by using a fluorescent quenching techniquethat measures the increase or decrease in fluorescence emissionintensity (excitation=295 nm; emission=340 nm, 16 nm band-pass) at 25°C. of a 20 nM anti-antigen antibody (Fab form) in PBS, pH 7.2, in thepresence of increasing concentrations of antigen as measured in aspectrometer, such as a stop-flow equipped spectrophometer (AvivInstruments) or a 8000-series SLM-AMINCO™ spectrophotometer(ThermoSpectronic) with a stirred cuvette.

(i) Antigen Preparation

Soluble antigens or fragments thereof, optionally conjugated to othermolecules, can be used as immunogens for generating antibodies. Fortransmembrane molecules, such as receptors, fragments of these (e.g. theextracellular domain of a receptor) can be used as the immunogen.Alternatively, cells expressing the transmembrane molecule can be usedas the immunogen. Such cells can be derived from a natural source (e.g.cancer cell lines) or may be cells which have been transformed byrecombinant techniques to express the transmembrane molecule. Otherantigens and forms thereof useful for preparing antibodies will beapparent to those in the art.

(ii) Certain Antibody-Based Methods

Polyclonal antibodies are preferably raised in animals by multiplesubcutaneous (sc) or intraperitoneal (ip) injections of the relevantantigen and an adjuvant. It may be useful to conjugate the relevantantigen to a protein that is immunogenic in the species to be immunized,e.g., keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, orsoybean trypsin inhibitor using a bifunctional or derivatizing agent,for example, maleimidobenzoyl sulfosuccinimide ester (conjugationthrough cysteine residues), N-hydroxysuccinimide (through lysineresidues), glutaraldehyde, succinic anhydride, SOCl₂, or R¹N═C═NR, whereR and R¹ are different alkyl groups.

Animals are immunized against the antigen, immunogenic conjugates, orderivatives by combining, e.g., 100 μg or 5 μg of the protein orconjugate (for rabbits or mice, respectively) with 3 volumes of Freund'scomplete adjuvant and injecting the solution intradermally at multiplesites. One month later the animals are boosted with 1/5 to 1/10 theoriginal amount of peptide or conjugate in Freund's complete adjuvant bysubcutaneous injection at multiple sites. Seven to 14 days later theanimals are bled and the serum is assayed for antibody titer. Animalsare boosted until the titer plateaus. Preferably, the animal is boostedwith the conjugate of the same antigen, but conjugated to a differentprotein and/or through a different cross-linking reagent. Conjugatesalso can be made in recombinant cell culture as protein fusions. Also,aggregating agents such as alum are suitably used to enhance the immuneresponse.

Monoclonal antibodies can be made using the hybridoma method firstdescribed by Kohler et al., Nature, 256:495 (1975), and furtherdescribed, e.g., in Hongo et al., Hybridoma, 14 (3): 253-260 (1995),Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring HarborLaboratory Press, 2nd ed. 1988); Hammerling et al., in: MonoclonalAntibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981), and Ni,Xiandai Mianyixue, 26(4):265-268 (2006) regarding human-humanhybridomas. Additional methods include those described, for example, inU.S. Pat. No. 7,189,826 regarding production of monoclonal human naturalIgM antibodies from hybridoma cell lines. Human hybridoma technology(Trioma technology) is described in Vollmers and Brandlein, Histologyand Histopathology, 20(3):927-937 (2005) and Vollmers and Brandlein,Methods and Findings in Experimental and Clinical Pharmacology,27(3):185-91 (2005).

For various other hybridoma techniques, see, e.g., US 2006/258841; US2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US2005/100546; US 2005/026229; and U.S. Pat. Nos. 7,078,492 and 7,153,507.An exemplary protocol for producing monoclonal antibodies using thehybridoma method is described as follows. In one embodiment, a mouse orother appropriate host animal, such as a hamster, is immunized to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the protein used for immunization. Antibodiesare raised in animals by multiple subcutaneous (sc) or intraperitoneal(ip) injections of a polypeptide of the invention or a fragment thereof,and an adjuvant, such as monophosphoryl lipid A (MPL)/trehalosedicrynomycolate (TDM) (Ribi Immunochem. Research, Inc., Hamilton,Mont.). A polypeptide of the invention (e.g., antigen) or a fragmentthereof may be prepared using methods well known in the art, such asrecombinant methods, some of which are further described herein. Serumfrom immunized animals is assayed for anti-antigen antibodies, andbooster immunizations are optionally administered. Lymphocytes fromanimals producing anti-antigen antibodies are isolated. Alternatively,lymphocytes may be immunized in vitro.

Lymphocytes are then fused with myeloma cells using a suitable fusingagent, such as polyethylene glycol, to form a hybridoma cell. See, e.g.,Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103(Academic Press, 1986). Myeloma cells may be used that fuse efficiently,support stable high-level production of antibody by the selectedantibody-producing cells, and are sensitive to a medium such as HATmedium. Exemplary myeloma cells include, but are not limited to, murinemyeloma lines, such as those derived from MOPC-21 and MPC-11 mousetumors available from the Salk Institute Cell Distribution Center, SanDiego, Calif. USA, and SP-2 or X63-Ag8-653 cells available from theAmerican Type Culture Collection, Rockville, Md. USA. Human myeloma andmouse-human heteromyeloma cell lines also have been described for theproduction of human monoclonal antibodies (Kozbor, J. Immunol., 133:3001(1984); Brodeur et al., Monoclonal Antibody Production Techniques andApplications, pp. 51-63 (Marcel Dekker, Inc., New York, 1987)).

The hybridoma cells thus prepared are seeded and grown in a suitableculture medium, e.g., a medium that contains one or more substances thatinhibit the growth or survival of the unfused, parental myeloma cells.For example, if the parental myeloma cells lack the enzyme hypoxanthineguanine phosphoribosyl transferase (HGPRT or HPRT), the culture mediumfor the hybridomas typically will include hypoxanthine, aminopterin, andthymidine (HAT medium), which substances prevent the growth ofHGPRT-deficient cells. Preferably, serum-free hybridoma cell culturemethods are used to reduce use of animal-derived serum such as fetalbovine serum, as described, for example, in Even et al., Trends inBiotechnology, 24(3), 105-108 (2006).

Oligopeptides as tools for improving productivity of hybridoma cellcultures are described in Franek, Trends in Monoclonal AntibodyResearch, 111-122 (2005). Specifically, standard culture media areenriched with certain amino acids (alanine, serine, asparagine,proline), or with protein hydrolyzate fractions, and apoptosis may besignificantly suppressed by synthetic oligopeptides, constituted ofthree to six amino acid residues. The peptides are present at millimolaror higher concentrations.

Culture medium in which hybridoma cells are growing may be assayed forproduction of monoclonal antibodies that bind to an antibody of theinvention. The binding specificity of monoclonal antibodies produced byhybridoma cells may be determined by immunoprecipitation or by an invitro binding assay, such as radioimmunoassay (RIA) or enzyme-linkedimmunoadsorbent assay (ELISA). The binding affinity of the monoclonalantibody can be determined, for example, by Scatchard analysis. See,e.g., Munson et al., Anal. Biochem., 107:220 (1980).

After hybridoma cells are identified that produce antibodies of thedesired specificity, affinity, and/or activity, the clones may besubcloned by limiting dilution procedures and grown by standard methods.See, e.g., Goding, supra. Suitable culture media for this purposeinclude, for example, D-MEM or RPMI-1640 medium. In addition, hybridomacells may be grown in vivo as ascites tumors in an animal. Monoclonalantibodies secreted by the subclones are suitably separated from theculture medium, ascites fluid, or serum by conventional immunoglobulinpurification procedures such as, for example, protein A-Sepharose,hydroxylapatite chromatography, gel electrophoresis, dialysis, oraffinity chromatography. One procedure for isolation of proteins fromhybridoma cells is described in US 2005/176122 and U.S. Pat. No.6,919,436. The method includes using minimal salts, such as lyotropicsalts, in the binding process and preferably also using small amounts oforganic solvents in the elution process.

(iii) Library-Derived Antibodies

Antibodies useful in the invention may be isolated by screeningcombinatorial libraries for antibodies with the desired activity oractivities. For example, a variety of methods are known in the art forgenerating phage display libraries and screening such libraries forantibodies possessing the desired binding characteristics. Additionalmethods are reviewed, e.g., in Hoogenboom et al. in Methods in MolecularBiology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., 2001)and further described, e.g., in the McCafferty et al., Nature348:552-554; Clackson et al., Nature 352: 624-628 (1991); Marks et al.,J. Mol. Biol. 222: 581-597 (1992); Marks and Bradbury, in Methods inMolecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, N.J.,2003); Sidhu et al., J. Mol. Biol. 338(2): 299-310 (2004); Lee et al.,J. Mol. Biol. 340(5): 1073-1093 (2004); Fellouse, Proc. Natl. Acad. Sci.USA 101(34): 12467-12472 (2004); and Lee et al., J. Immunol. Methods284(1-2): 119-132(2004).

In certain phage display methods, repertoires of VH and VL genes areseparately cloned by polymerase chain reaction (PCR) and recombinedrandomly in phage libraries, which can then be screened forantigen-binding phage as described in Winter et al., Ann. Rev. Immunol.,12: 433-455 (1994). Phage typically display antibody fragments, eitheras single-chain Fv (scFv) fragments or as Fab fragments. Libraries fromimmunized sources provide high-affinity antibodies to the immunogenwithout the requirement of constructing hybridomas. Alternatively, thenaive repertoire can be cloned (e.g., from human) to provide a singlesource of antibodies to a wide range of non-self and also self-antigenswithout any immunization as described by Griffiths et al., EMBO J, 12:725-734 (1993). Finally, naive libraries can also be made syntheticallyby cloning unrearranged V-gene segments from stem cells, and using PCRprimers containing random sequence to encode the highly variable CDR3regions and to accomplish rearrangement in vitro, as described byHoogenboom and Winter, J. Mol. Biol., 227: 381-388 (1992). Patentpublications describing human antibody phage libraries include, forexample: U.S. Pat. No. 5,750,373, and US Patent Publication Nos.2005/0079574, 2005/0119455, 2005/0266000, 2007/0117126, 2007/0160598,2007/0237764, 2007/0292936, and 2009/0002360.

Antibodies or antibody fragments isolated from human antibody librariesare considered human antibodies or human antibody fragments herein.

(iv) Chimeric, Humanized and Human Antibodies

In certain embodiments, an antibody provided herein is a chimericantibody. Certain chimeric antibodies are described, e.g., in U.S. Pat.No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). In one example, a chimeric antibody comprises anon-human variable region (e.g., a variable region derived from a mouse,rat, hamster, rabbit, or non-human primate, such as a monkey) and ahuman constant region. In a further example, a chimeric antibody is a“class switched” antibody in which the class or subclass has beenchanged from that of the parent antibody. Chimeric antibodies includeantigen-binding fragments thereof.

In certain embodiments, a chimeric antibody is a humanized antibody.Typically, a non-human antibody is humanized to reduce immunogenicity tohumans, while retaining the specificity and affinity of the parentalnon-human antibody. Generally, a humanized antibody comprises one ormore variable domains in which HVRs, e.g., CDRs, (or portions thereof)are derived from a non-human antibody, and FRs (or portions thereof) arederived from human antibody sequences. A humanized antibody optionallywill also comprise at least a portion of a human constant region. Insome embodiments, some FR residues in a humanized antibody aresubstituted with corresponding residues from a non-human antibody (e.g.,the antibody from which the HVR residues are derived), e.g., to restoreor improve antibody specificity or affinity.

Humanized antibodies and methods of making them are reviewed, e.g., inAlmagro and Fransson, Front. Biosci. 13:1619-1633 (2008), and arefurther described, e.g., in Riechmann et al., Nature 332:323-329 (1988);Queen et al., Proc. Nat'l Acad. Sci. USA 86:10029-10033 (1989); U.S.Pat. Nos. 5,821,337, 7,527,791, 6,982,321, and 7,087,409; Kashmiri etal., Methods 36:25-34 (2005) (describing SDR (a-CDR) grafting); Padlan,Mol. Immunol. 28:489-498 (1991) (describing “resurfacing”); Dall'Acquaet al., Methods 36:43-60 (2005) (describing “FR shuffling”); and Osbournet al., Methods 36:61-68 (2005) and Klimka et al., Br. J. Cancer,83:252-260 (2000) (describing the “guided selection” approach to FRshuffling).

Human framework regions that may be used for humanization include butare not limited to: framework regions selected using the “best-fit”method (see, e.g., Sims et al. J. Immunol. 151:2296 (1993)); frameworkregions derived from the consensus sequence of human antibodies of aparticular subgroup of light or heavy chain variable regions (see, e.g.,Carter et al. Proc. Natl. Acad. Sci. USA, 89:4285 (1992); and Presta etal. J. Immunol., 151:2623 (1993)); human mature (somatically mutated)framework regions or human germline framework regions (see, e.g.,Almagro and Fransson, Front. Biosci. 13:1619-1633 (2008)); and frameworkregions derived from screening FR libraries (see, e.g., Baca et al., J.Biol. Chem. 272:10678-10684 (1997) and Rosok et al., J. Biol. Chem.271:22611-22618 (1996)).

In certain embodiments, an antibody provided herein is a human antibody.Human antibodies can be produced using various techniques known in theart. Human antibodies are described generally in van Dijk and van deWinkel, Curr. Opin. Pharmacol. 5: 368-74 (2001) and Lonberg, Curr. Opin.Immunol. 20:450-459 (2008).

Human antibodies may be prepared by administering an immunogen to atransgenic animal that has been modified to produce intact humanantibodies or intact antibodies with human variable regions in responseto antigenic challenge. Such animals typically contain all or a portionof the human immunoglobulin loci, which replace the endogenousimmunoglobulin loci, or which are present extrachromosomally orintegrated randomly into the animal's chromosomes. In such transgenicmice, the endogenous immunoglobulin loci have generally beeninactivated. For review of methods for obtaining human antibodies fromtransgenic animals, see Lonberg, Nat. Biotech. 23:1117-1125 (2005). Seealso, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 describing XENOMOUSE™technology; U.S. Pat. No. 5,770,429 describing HuMAB® technology; U.S.Pat. No. 7,041,870 describing K-M MOUSE® technology, and U.S. PatentApplication Publication No. US 2007/0061900, describing VELocIMousE®technology). Human variable regions from intact antibodies generated bysuch animals may be further modified, e.g., by combining with adifferent human constant region.

Human antibodies can also be made by hybridoma-based methods. Humanmyeloma and mouse-human heteromyeloma cell lines for the production ofhuman monoclonal antibodies have been described. (See, e.g., Kozbor J.Immunol., 133: 3001 (1984); Brodeur et al., Monoclonal AntibodyProduction Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc.,New York, 1987); and Boerner et al., J. Immunol., 147: 86 (1991).) Humanantibodies generated via human B-cell hybridoma technology are alsodescribed in Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562(2006). Additional methods include those described, for example, in U.S.Pat. No. 7,189,826 (describing production of monoclonal human IgMantibodies from hybridoma cell lines) and Ni, Xiandai Mianyixue,26(4):265-268 (2006) (describing human-human hybridomas). Humanhybridoma technology (Trioma technology) is also described in Vollmersand Brandlein, Histology and Histopathology, 20(3):927-937 (2005) andVollmers and Brandlein, Methods and Findings in Experimental andClinical Pharmacology, 27(3):185-91 (2005).

Human antibodies may also be generated by isolating Fv clone variabledomain sequences selected from human-derived phage display libraries.Such variable domain sequences may then be combined with a desired humanconstant domain. Techniques for selecting human antibodies from antibodylibraries are described below.

(v) Antibody Fragments

Antibody fragments may be generated by traditional means, such asenzymatic digestion, or by recombinant techniques. In certaincircumstances there are advantages of using antibody fragments, ratherthan whole antibodies. The smaller size of the fragments allows forrapid clearance, and may lead to improved access to solid tumors. For areview of certain antibody fragments, see Hudson et al. (2003) Nat. Med.9:129-134.

Various techniques have been developed for the production of antibodyfragments. Traditionally, these fragments were derived via proteolyticdigestion of intact antibodies (see, e.g., Morimoto et al., Journal ofBiochemical and Biophysical Methods 24:107-117 (1992); and Brennan etal., Science, 229:81 (1985)). However, these fragments can now beproduced directly by recombinant host cells. Fab, Fv and ScFv antibodyfragments can all be expressed in and secreted from E. coli, thusallowing the facile production of large amounts of these fragments.Antibody fragments can be isolated from the antibody phage librariesdiscussed above. Alternatively, Fab′-SH fragments can be directlyrecovered from E. coli and chemically coupled to form F(ab′)₂ fragments(Carter et al., Bio/Technology 10:163-167 (1992)). According to anotherapproach, F(ab′)₂ fragments can be isolated directly from recombinanthost cell culture. Fab and F(ab′)₂ fragment with increased in vivohalf-life comprising salvage receptor binding epitope residues aredescribed in U.S. Pat. No. 5,869,046. Other techniques for theproduction of antibody fragments will be apparent to the skilledpractitioner. In certain embodiments, an antibody is a single chain Fvfragment (scFv). See WO 93/16185; U.S. Pat. Nos. 5,571,894; and5,587,458. Fv and scFv are the only species with intact combining sitesthat are devoid of constant regions; thus, they may be suitable forreduced nonspecific binding during in vivo use. scFv fusion proteins maybe constructed to yield fusion of an effector protein at either theamino or the carboxy terminus of an scFv. See Antibody Engineering, ed.Borrebaeck, supra. The antibody fragment may also be a “linearantibody”, e.g., as described in U.S. Pat. No. 5,641,870, for example.Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific Antibodies

Multispecific antibodies have binding specificities for at least twodifferent epitopes, where the epitopes are usually from differentantigens. While such molecules normally will only bind two differentepitopes (i.e. bispecific antibodies, BsAbs), antibodies with additionalspecificities such as trispecific antibodies are encompassed by thisexpression when used herein. Bispecific antibodies can be prepared asfull length antibodies or antibody fragments (e.g. F(ab′)₂ bispecificantibodies).

Methods for making bispecific antibodies are known in the art.Traditional production of full length bispecific antibodies is based onthe coexpression of two immunoglobulin heavy chain-light chain pairs,where the two chains have different specificities (Millstein et al.,Nature, 305:537-539 (1983)). Because of the random assortment ofimmunoglobulin heavy and light chains, these hybridomas (quadromas)produce a potential mixture of 10 different antibody molecules, of whichonly one has the correct bispecific structure. Purification of thecorrect molecule, which is usually done by affinity chromatographysteps, is rather cumbersome, and the product yields are low. Similarprocedures are disclosed in WO 93/08829, and in Traunecker et al., EMBOJ., 10:3655-3659 (1991).

One approach known in the art for making bispecific antibodies is the“knobs-into-holes” or “protuberance-into-cavity” approach (see, e.g.,U.S. Pat. No. 5,731,168). In this approach, two immunoglobulinpolypeptides (e.g., heavy chain polypeptides) each comprise aninterface. An interface of one immunoglobulin polypeptide interacts witha corresponding interface on the other immunoglobulin polypeptide,thereby allowing the two immunoglobulin polypeptides to associate. Theseinterfaces may be engineered such that a “knob” or “protuberance” (theseterms may be used interchangeably herein) located in the interface ofone immunoglobulin polypeptide corresponds with a “hole” or “cavity”(these terms may be used interchangeably herein) located in theinterface of the other immunoglobulin polypeptide. In some embodiments,the hole is of identical or similar size to the knob and suitablypositioned such that when the two interfaces interact, the knob of oneinterface is positionable in the corresponding hole of the otherinterface. Without wishing to be bound to theory, this is thought tostabilize the heteromultimer and favor formation of the heteromultimerover other species, for example homomultimers. In some embodiments, thisapproach may be used to promote the heteromultimerization of twodifferent immunoglobulin polypeptides, creating a bispecific antibodycomprising two immunoglobulin polypeptides with binding specificitiesfor different epitopes.

In some embodiments, a knob may be constructed by replacing a smallamino acid side chain with a larger side chain. In some embodiments, ahole may be constructed by replacing a large amino acid side chain witha smaller side chain. Knobs or holes may exist in the originalinterface, or they may be introduced synthetically. For example, knobsor holes may be introduced synthetically by altering the nucleic acidsequence encoding the interface to replace at least one “original” aminoacid residue with at least one “import” amino acid residue. Methods foraltering nucleic acid sequences may include standard molecular biologytechniques well known in the art. The side chain volumes of variousamino acid residues are shown in the following table. In someembodiments, original residues have a small side chain volume (e.g.,alanine, asparagine, aspartic acid, glycine, serine, threonine, orvaline), and import residues for forming a knob are naturally occurringamino acids and may include arginine, phenylalanine, tyrosine, andtryptophan. In some embodiments, original residues have a large sidechain volume (e.g., arginine, phenylalanine, tyrosine, and tryptophan),and import residues for forming a hole are naturally occurring aminoacids and may include alanine, serine, threonine, and valine.

TABLE 1 Properties of amino acid residues Accessible surface One-letterMass^(a) Volume^(b) area^(c) Amino acid abbreviation (daltons) (Å³) (Å²)Alanine (Ala) A 71.08 88.6 115 Arginine (Arg) R 156.20 173.4 225Asparagine (Asn) N 114.11 117.7 160 Aspartic Acid (Asp) D 115.09 111.1150 Cysteine (Cys) C 103.14 108.5 135 Glutamine (Gln) Q 128.14 143.9 180Glutamic Acid (Glu) E 129.12 138.4 190 Glycine (Gly) G 57.06 60.1 75Histidine (His) H 137.15 153.2 195 Isoleucine (Be) I 113.17 166.7 175Leucine (Leu) L 113.17 166.7 170 Lysine (Lys) K 128.18 168.6 200Methionine (Met) M 131.21 162.9 185 Phenylalanine (Phe) F 147.18 189.9210 Proline (Pro) P 97.12 122.7 145 Serine (Ser) S 87.08 89.0 115Threonine (Thr) T 101.11 116.1 140 Tryptophan (Trp) W 186.21 227.8 255Tyrosine (Tyr) Y 163.18 193.6 230 Valine (Val) V 99.14 140.0 155^(a)Molecular weight of amino acid minus that of water. Values fromHandbook of Chemistry and Physics, 43^(rd) ed. Cleveland, ChemicalRubber Publishing Co., 1961. ^(b)Values from A.A. Zamyatnin, Prog.Biophys. Mol. Biol. 24:107-123, 1972. ^(c)Values from C. Chothia, J.Mol. Biol. 105:1-14, 1975. The accessible surface area is defined inFIGS. 6-20 of this reference.

In some embodiments, original residues for forming a knob or hole areidentified based on the three-dimensional structure of theheteromultimer. Techniques known in the art for obtaining athree-dimensional structure may include X-ray crystallography and NMR.In some embodiments, the interface is the CH3 domain of animmunoglobulin constant domain. In these embodiments, the CH3/CH3interface of human IgG₁ involves sixteen residues on each domain locatedon four anti-parallel β-strands. Without wishing to be bound to theory,mutated residues are preferably located on the two central anti-parallelβ-strands to minimize the risk that knobs can be accommodated by thesurrounding solvent, rather than the compensatory holes in the partnerCH3 domain. In some embodiments, the mutations forming correspondingknobs and holes in two immunoglobulin polypeptides correspond to one ormore pairs provided in the following table.

TABLE 2 Exemplary sets of corresponding knob-and hole-forming mutationsCH3 of first immunoglobulin CH3 of second immunoglobulin T366Y Y407TT366W Y407A F405A T394W Y407T T366Y T366Y:F405A T394W:Y407T T366W:F405WT394S:Y407A F405W:Y407A T366W:T394S F405W T394S Mutations are denoted bythe original residue, followed by the position using the EU numberingsystem, and then the import residue (all residues are given insingle-letter amino acid code). Multiple mutations are separated by acolon.

In some embodiments, an immunoglobulin polypeptide comprises a CH3domain comprising one or more amino acid substitutions listed in Table 2above. In some embodiments, a bispecific antibody comprises a firstimmunoglobulin polypeptide comprising a CH3 domain comprising one ormore amino acid substitutions listed in the left column of Table 2, anda second immunoglobulin polypeptide comprising a CH3 domain comprisingone or more corresponding amino acid substitutions listed in the rightcolumn of Table 2.

Following mutation of the DNA as discussed above, polynucleotidesencoding modified immunoglobulin polypeptides with one or morecorresponding knob- or hole-forming mutations may be expressed andpurified using standard recombinant techniques and cell systems known inthe art. See, e.g., U.S. Pat. Nos. 5,731,168; 5,807,706; 5,821,333;7,642,228; 7,695,936; 8,216,805; U.S. Pub. No. 2013/0089553; and Spiesset al., Nature Biotechnology 31: 753-758, 2013. Modified immunoglobulinpolypeptides may be produced using prokaryotic host cells, such as E.coli, or eukaryotic host cells, such as CHO cells. Corresponding knob-and hole-bearing immunoglobulin polypeptides may be expressed in hostcells in co-culture and purified together as a heteromultimer, or theymay be expressed in single cultures, separately purified, and assembledin vitro. In some embodiments, two strains of bacterial host cells (oneexpressing an immunoglobulin polypeptide with a knob, and the otherexpressing an immunoglobulin polypeptide with a hole) are co-culturedusing standard bacterial culturing techniques known in the art. In someembodiments, the two strains may be mixed in a specific ratio, e.g., soas to achieve equal expression levels in culture. In some embodiments,the two strains may be mixed in a 50:50, 60:40, or 70:30 ratio. Afterpolypeptide expression, the cells may be lysed together, and protein maybe extracted. Standard techniques known in the art that allow formeasuring the abundance of homo-multimeric vs. hetero-multimeric speciesmay include size exclusion chromatography. In some embodiments, eachmodified immunoglobulin polypeptide is expressed separately usingstandard recombinant techniques, and they may be assembled together invitro. Assembly may be achieved, for example, by purifying each modifiedimmunoglobulin polypeptide, mixing and incubating them together in equalmass, reducing disulfides (e.g., by treating with dithiothreitol),concentrating, and reoxidizing the polypeptides. Formed bispecificantibodies may be purified using standard techniques includingcation-exchange chromatography and measured using standard techniquesincluding size exclusion chromatography. For a more detailed descriptionof these methods, see Spiess et al., Nat Biotechnol 31:753-8, 2013. Insome embodiments, modified immunoglobulin polypeptides may be expressedseparately in CHO cells and assembled in vitro using the methodsdescribed above.

According to a different approach, antibody variable domains with thedesired binding specificities (antibody-antigen combining sites) arefused to immunoglobulin constant domain sequences. The fusion preferablyis with an immunoglobulin heavy chain constant domain, comprising atleast part of the hinge, CH2, and CH3 regions. It is typical to have thefirst heavy-chain constant region (CH1) containing the site necessaryfor light chain binding, present in at least one of the fusions. DNAsencoding the immunoglobulin heavy chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Thisprovides for great flexibility in adjusting the mutual proportions ofthe three polypeptide fragments in embodiments when unequal ratios ofthe three polypeptide chains used in the construction provide theoptimum yields. It is, however, possible to insert the coding sequencesfor two or all three polypeptide chains in one expression vector whenthe expression of at least two polypeptide chains in equal ratiosresults in high yields or when the ratios are of no particularsignificance.

In one embodiment of this approach, the bispecific antibodies arecomposed of a hybrid immunoglobulin heavy chain with a first bindingspecificity in one arm, and a hybrid immunoglobulin heavy chain-lightchain pair (providing a second binding specificity) in the other arm. Itwas found that this asymmetric structure facilitates the separation ofthe desired bispecific compound from unwanted immunoglobulin chaincombinations, as the presence of an immunoglobulin light chain in onlyone half of the bispecific molecule provides for a facile way ofseparation. This approach is disclosed in WO 94/04690. For furtherdetails of generating bispecific antibodies see, for example, Suresh etal., Methods in Enzymology, 121:210 (1986).

According to another approach described in WO96/27011, the interfacebetween a pair of antibody molecules can be engineered to maximize thepercentage of heterodimers which are recovered from recombinant cellculture. One interface comprises at least a part of the C_(H) 3 domainof an antibody constant domain. In this method, one or more small aminoacid side chains from the interface of the first antibody molecule arereplaced with larger side chains (e.g. tyrosine or tryptophan).Compensatory “cavities” of identical or similar size to the large sidechain(s) are created on the interface of the second antibody molecule byreplacing large amino acid side chains with smaller ones (e.g. alanineor threonine). This provides a mechanism for increasing the yield of theheterodimer over other unwanted end-products such as homodimers.

Bispecific antibodies include cross-linked or “heteroconjugate”antibodies. For example, one of the antibodies in the heteroconjugatecan be coupled to avidin, the other to biotin. Such antibodies have, forexample, been proposed to target immune system cells to unwanted cells(U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO91/00360, WO 92/200373, and EP 03089). Heteroconjugate antibodies may bemade using any convenient cross-linking methods. Suitable cross-linkingagents are well known in the art, and are disclosed in U.S. Pat. No.4,676,980, along with a number of cross-linking techniques.

Techniques for generating bispecific antibodies from antibody fragmentshave also been described in the literature. For example, bispecificantibodies can be prepared using chemical linkage. Brennan et al.,Science, 229: 81 (1985) describe a procedure wherein intact antibodiesare proteolytically cleaved to generate F(ab′)2 fragments. Thesefragments are reduced in the presence of the dithiol complexing agentsodium arsenite to stabilize vicinal dithiols and prevent intermoleculardisulfide formation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

Recent progress has facilitated the direct recovery of Fab′-SH fragmentsfrom E. coli, which can be chemically coupled to form bispecificantibodies. Shalaby et al., J. Exp. Med., 175: 217-225 (1992) describethe production of a fully humanized bispecific antibody F(ab′)2molecule. Each Fab′ fragment was separately secreted from E. coli andsubjected to directed chemical coupling in vitro to form the bispecificantibody.

Various techniques for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA, 90:6444-6448 (1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See Gruber et al, J. Immunol, 152:5368 (1994).

Another technique for making bispecific antibody fragments is the“bispecific T cell engager” or BiTE® approach (see, e.g., WO2004/106381,WO2005/061547, WO2007/042261, and WO2008/119567). This approach utilizestwo antibody variable domains arranged on a single polypeptide. Forexample, a single polypeptide chain includes two single chain Fv (scFv)fragments, each having a variable heavy chain (V_(H)) and a variablelight chain (V_(L)) domain separated by a polypeptide linker of a lengthsufficient to allow intramolecular association between the two domains.This single polypeptide further includes a polypeptide spacer sequencebetween the two scFv fragments. Each scFv recognizes a differentepitope, and these epitopes may be specific for different cell types,such that cells of two different cell types are brought into closeproximity or tethered when each scFv is engaged with its cognateepitope. One particular embodiment of this approach includes a scFvrecognizing a cell-surface antigen expressed by an immune cell, e.g., aCD3 polypeptide on a T cell, linked to another scFv that recognizes acell-surface antigen expressed by a target cell, such as a malignant ortumor cell.

As it is a single polypeptide, the bispecific T cell engager may beexpressed using any prokaryotic or eukaryotic cell expression systemknown in the art, e.g., a CHO cell line. However, specific purificationtechniques (see, e.g., EP1691833) may be necessary to separate monomericbispecific T cell engagers from other multimeric species, which may havebiological activities other than the intended activity of the monomer.In one exemplary purification scheme, a solution containing secretedpolypeptides is first subjected to a metal affinity chromatography, andpolypeptides are eluted with a gradient of imidazole concentrations.This eluate is further purified using anion exchange chromatography, andpolypeptides are eluted using with a gradient of sodium chlorideconcentrations. Finally, this eluate is subjected to size exclusionchromatography to separate monomers from multimeric species.

Antibodies with more than two valencies are contemplated. For example,trispecific antibodies can be prepared. Tuft et al. J. Immunol. 147: 60(1991).

(vii) Single-Domain Antibodies

A single-domain antibody is a single polypeptide chain comprising all ora portion of the heavy chain variable domain or all or a portion of thelight chain variable domain of an antibody. In certain embodiments, asingle-domain antibody is a human single-domain antibody (Domantis,Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516 BI). In oneembodiment, a single-domain antibody consists of all or a portion of theheavy chain variable domain of an antibody.

(viii) Antibody Variants

In some embodiments, amino acid sequence modification(s) of theantibodies described herein are contemplated. For example, it may bedesirable to improve the binding affinity and/or other biologicalproperties of the antibody. Amino acid sequence variants of the antibodymay be prepared by introducing appropriate changes into the nucleotidesequence encoding the antibody, or by peptide synthesis. Suchmodifications include, for example, deletions from, and/or insertionsinto and/or substitutions of, residues within the amino acid sequencesof the antibody. Any combination of deletion, insertion, andsubstitution can be made to arrive at the final construct, provided thatthe final construct possesses the desired characteristics. The aminoacid alterations may be introduced in the subject antibody amino acidsequence at the time that sequence is made.

(ix) Substitution, Insertion, and Deletion Variants

In certain embodiments, antibody variants having one or more amino acidsubstitutions are provided. Sites of interest for substitutionalmutagenesis include the HVRs and FRs. Conservative substitutions areshown in Table 1 under the heading of “preferred substitutions.” Moresubstantial changes are provided in Table 1 under the heading of“exemplary substitutions,” and as further described below in referenceto amino acid side chain classes. Amino acid substitutions may beintroduced into an antibody of interest and the products screened for adesired activity, e.g., retained/improved antigen binding, decreasedimmunogenicity, or improved ADCC or CDC.

TABLE 3 Exemplary Substitutions. Original Preferred Residue ExemplarySubstitutions Substitutions Ala (A) Val; Leu; Ile Val Arg (R) Lys; Gln;Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp (D) Glu; Asn Glu Cys (C)Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp; Gln Asp Gly (G) Ala AlaHis (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phe;Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys (K)Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp; Leu; Val; Ile;Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile; Leu; Met;Phe; Ala; Norleucine Leu

Amino acids may be grouped according to common side-chain properties:

a. hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;b. neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;c. acidic: Asp, Glu;d. basic: His, Lys, Arg;e. residues that influence chain orientation: Gly, Pro;f. aromatic: Trp, Tyr, Phe.

Non-conservative substitutions will entail exchanging a member of one ofthese classes for another class.

One type of substitutional variant involves substituting one or morehypervariable region residues of a parent antibody (e.g. a humanized orhuman antibody). Generally, the resulting variant(s) selected forfurther study will have modifications (e.g., improvements) in certainbiological properties (e.g., increased affinity, reduced immunogenicity)relative to the parent antibody and/or will have substantially retainedcertain biological properties of the parent antibody. An exemplarysubstitutional variant is an affinity matured antibody, which may beconveniently generated, e.g., using phage display-based affinitymaturation techniques such as those described herein. Briefly, one ormore HVR residues are mutated and the variant antibodies displayed onphage and screened for a particular biological activity (e.g. bindingaffinity).

Alterations (e.g., substitutions) may be made in HVRs, e.g., to improveantibody affinity. Such alterations may be made in HVR “hotspots,” i.e.,residues encoded by codons that undergo mutation at high frequencyduring the somatic maturation process (see, e.g., Chowdhury, MethodsMol. Biol. 207:179-196 (2008)), and/or SDRs (a-CDRs), with the resultingvariant VH or VL being tested for binding affinity. Affinity maturationby constructing and reselecting from secondary libraries has beendescribed, e.g., in Hoogenboom et al. in Methods in Molecular Biology178:1-37 (O'Brien et al., ed., Human Press, Totowa, N.J., (2001).) Insome embodiments of affinity maturation, diversity is introduced intothe variable genes chosen for maturation by any of a variety of methods(e.g., error-prone PCR, chain shuffling, or oligonucleotide-directedmutagenesis). A secondary library is then created. The library is thenscreened to identify any antibody variants with the desired affinity.Another method to introduce diversity involves HVR-directed approaches,in which several HVR residues (e.g., 4-6 residues at a time) arerandomized. HVR residues involved in antigen binding may be specificallyidentified, e.g., using alanine scanning mutagenesis or modeling. CDR-H3and CDR-L3 in particular are often targeted.

In certain embodiments, substitutions, insertions, or deletions mayoccur within one or more HVRs so long as such alterations do notsubstantially reduce the ability of the antibody to bind antigen. Forexample, conservative alterations (e.g., conservative substitutions asprovided herein) that do not substantially reduce binding affinity maybe made in HVRs. Such alterations may be outside of HVR “hotspots” orSDRs. In certain embodiments of the variant VH and VL sequences providedabove, each HVR either is unaltered, or contains no more than one, twoor three amino acid substitutions.

A useful method for identification of residues or regions of an antibodythat may be targeted for mutagenesis is called “alanine scanningmutagenesis” as described by Cunningham and Wells (1989) Science,244:1081-1085. In this method, a residue or group of target residues(e.g., charged residues such as arg, asp, his, lys, and glu) areidentified and replaced by a neutral or negatively charged amino acid(e.g., alanine or polyalanine) to determine whether the interaction ofthe antibody with antigen is affected. Further substitutions may beintroduced at the amino acid locations demonstrating functionalsensitivity to the initial substitutions. Alternatively, oradditionally, a crystal structure of an antigen-antibody complex toidentify contact points between the antibody and antigen. Such contactresidues and neighboring residues may be targeted or eliminated ascandidates for substitution. Variants may be screened to determinewhether they contain the desired properties.

Amino acid sequence insertions include amino- and/or carboxyl-terminalfusions ranging in length from one residue to polypeptides containing ahundred or more residues, as well as intrasequence insertions of singleor multiple amino acid residues. Examples of terminal insertions includean antibody with an N-terminal methionyl residue. Other insertionalvariants of the antibody molecule include the fusion to the N- orC-terminus of the antibody to an enzyme (e.g., for ADEPT) or apolypeptide which increases the serum half-life of the antibody.

(x) Glycosylation Variants

In certain embodiments, an antibody provided herein is altered toincrease or decrease the extent to which the antibody is glycosylated.Addition or deletion of glycosylation sites to an antibody may beconveniently accomplished by altering the amino acid sequence such thatone or more glycosylation sites is created or removed.

Where the antibody comprises an Fc region, the carbohydrate attachedthereto may be altered. Native antibodies produced by mammalian cellstypically comprise a branched, biantennary oligosaccharide that isgenerally attached by an N-linkage to Asn297 of the CH2 domain of the Fcregion. See, e.g., Wright et al. TIBTECH 15:26-32 (1997). Theoligosaccharide may include various carbohydrates, e.g., mannose,N-acetyl glucosamine (GlcNAc), galactose, and sialic acid, as well as afucose attached to a GlcNAc in the “stem” of the biantennaryoligosaccharide structure. In some embodiments, modifications of theoligosaccharide in an antibody of the invention may be made in order tocreate antibody variants with certain improved properties.

In one embodiment, antibody variants are provided comprising an Fcregion wherein a carbohydrate structure attached to the Fc region hasreduced fucose or lacks fucose, which may improve ADCC function.Specifically, antibodies are contemplated herein that have reducedfucose relative to the amount of fucose on the same antibody produced ina wild-type CHO cell. That is, they are characterized by having a loweramount of fucose than they would otherwise have if produced by nativeCHO cells (e.g., a CHO cell that produce a native glycosylation pattern,such as, a CHO cell containing a native FUT8 gene). In certainembodiments, the antibody is one wherein less than about 50%, 40%, 30%,20%, 10%, or 5% of the N-linked glycans thereon comprise fucose. Forexample, the amount of fucose in such an antibody may be from 1% to 80%,from 1% to 65%, from 5% to 65% or from 20% to 40%. In certainembodiments, the antibody is one wherein none of the N-linked glycansthereon comprise fucose, i.e., wherein the antibody is completelywithout fucose, or has no fucose or is afucosylated. The amount offucose is determined by calculating the average amount of fucose withinthe sugar chain at Asn297, relative to the sum of all glycostructuresattached to Asn 297 (e. g. complex, hybrid and high mannose structures)as measured by MALDI-TOF mass spectrometry, as described in WO2008/077546, for example. Asn297 refers to the asparagine residuelocated at about position 297 in the Fc region (Eu numbering of Fcregion residues); however, Asn297 may also be located about ±3 aminoacids upstream or downstream of position 297, i.e., between positions294 and 300, due to minor sequence variations in antibodies. Suchfucosylation variants may have improved ADCC function. See, e.g., USPatent Publication Nos. US 2003/0157108 (Presta, L.); US 2004/0093621(Kyowa Hakko Kogyo Co., Ltd). Examples of publications related to“defucosylated” or “fucose-deficient” antibody variants include: US2003/0157108; WO 2000/61739; WO 2001/29246; US 2003/0115614; US2002/0164328; US 2004/0093621; US 2004/0132140; US 2004/0110704; US2004/0110282; US 2004/0109865; WO 2003/085119; WO 2003/084570; WO2005/035586; WO 2005/035778; WO2005/053742; WO2002/031140; Okazaki etal. J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al. Biotech.Bioeng. 87: 614 (2004). Examples of cell lines capable of producingdefucosylated antibodies include Lec13 CHO cells deficient in proteinfucosylation (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986);US Pat Appl No US 2003/0157108 A1, Presta, L; and WO 2004/056312 A1,Adams et al., especially at Example 11), and knockout cell lines, suchas alpha-1,6-fucosyltransferase gene, FUT8, knockout CHO cells (see,e.g., Yamane-Ohnuki et al. Biotech. Bioeng. 87: 614 (2004); Kanda, Y. etal., Biotechnol. Bioeng., 94(4):680-688 (2006); and WO2003/085107).

Antibody variants are further provided with bisected oligosaccharides,e.g., in which a biantennary oligosaccharide attached to the Fc regionof the antibody is bisected by GlcNAc. Such antibody variants may havereduced fucosylation and/or improved ADCC function. Examples of suchantibody variants are described, e.g., in WO 2003/011878 (Jean-Mairet etal.); U.S. Pat. No. 6,602,684 (Umana et al.); US 2005/0123546 (Umana etal.), and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Antibody variants with at least one galactose residue inthe oligosaccharide attached to the Fc region are also provided. Suchantibody variants may have improved CDC function. Such antibody variantsare described, e.g., in WO 1997/30087 (Patel et al.); WO 1998/58964(Raju, S.); and WO 1999/22764 (Raju, S.).

In certain embodiments, the antibody variants comprising an Fc regiondescribed herein are capable of binding to an FcγRIII In certainembodiments, the antibody variants comprising an Fc region describedherein have ADCC activity in the presence of human effector cells orhave increased ADCC activity in the presence of human effector cellscompared to the otherwise same antibody comprising a human wild-typeIgG1Fc region.

(xi) Fc Region Variants

In certain embodiments, one or more amino acid modifications may beintroduced into the Fc region of an antibody provided herein, therebygenerating an Fc region variant. The Fc region variant may comprise ahuman Fc region sequence (e.g., a human IgG1, IgG2, IgG3 or IgG4 Fcregion) comprising an amino acid modification (e.g. a substitution) atone or more amino acid positions.

In certain embodiments, the invention contemplates an antibody variantthat possesses some but not all effector functions, which make it adesirable candidate for applications in which the half life of theantibody in vivo is important yet certain effector functions (such ascomplement and ADCC) are unnecessary or deleterious. In vitro and/or invivo cytotoxicity assays can be conducted to confirm thereduction/depletion of CDC and/or ADCC activities. For example, Fcreceptor (FcR) binding assays can be conducted to ensure that theantibody lacks FcγR binding (hence likely lacking ADCC activity), butretains FcRn binding ability. The primary cells for mediating ADCC, NKcells, express Fc(RIII only, whereas monocytes express Fc(RI, Fc(RII andFc(RIII FcR expression on hematopoietic cells is summarized in Table 3on page 464 of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-492 (1991).Non-limiting examples of in vitro assays to assess ADCC activity of amolecule of interest is described in U.S. Pat. No. 5,500,362 (see, e.g.Hellstrom, I. et al. Proc. Nat'l Acad. Sci. USA 83:7059-7063 (1986)) andHellstrom, I et al., Proc. Nat'l Acad. Sci. USA 82:1499-1502 (1985);5,821,337 (see Bruggemann, M. et al., J. Exp. Med. 166:1351-1361(1987)). Alternatively, non-radioactive assays methods may be employed(see, for example, ACTI™ non-radioactive cytotoxicity assay for flowcytometry (CellTechnology, Inc. Mountain View, Calif.; and CytoTox 96®non-radioactive cytotoxicity assay (Promega, Madison, Wis.). Usefuleffector cells for such assays include peripheral blood mononuclearcells (PBMC) and Natural Killer (NK) cells. Alternatively, oradditionally, ADCC activity of the molecule of interest may be assessedin vivo, e.g., in an animal model such as that disclosed in Clynes etal. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). C1q binding assays mayalso be carried out to confirm that the antibody is unable to bind C1qand hence lacks CDC activity. See, e.g., C1q and C3c binding ELISA in WO2006/029879 and WO 2005/100402. To assess complement activation, a CDCassay may be performed (see, for example, Gazzano-Santoro et al., J.Immunol. Methods 202:163 (1996); Cragg, M. S. et al., Blood101:1045-1052 (2003); and Cragg, M. S. and M. J. Glennie, Blood103:2738-2743 (2004)). FcRn binding and in vivo clearance/half lifedeterminations can also be performed using methods known in the art(see, e.g., Petkova, S. B. et al., Int'l. Immunol. 18(12):1759-1769(2006)).

Antibodies with reduced effector function include those withsubstitution of one or more of Fc region residues 238, 265, 269, 270,297, 327 and 329 (U.S. Pat. No. 6,737,056). Such Fc mutants include Fcmutants with substitutions at two or more of amino acid positions 265,269, 270, 297 and 327, including the so-called “DANA” Fc mutant withsubstitution of residues 265 and 297 to alanine (U.S. Pat. No.7,332,581).

Certain antibody variants with improved or diminished binding to FcRsare described. (See, e.g., U.S. Pat. No. 6,737,056; WO 2004/056312, andShields et al., J. Biol. Chem. 9(2): 6591-6604 (2001).)

In certain embodiments, an antibody variant comprises an Fc region withone or more amino acid substitutions which improve ADCC, e.g.,substitutions at positions 298, 333, and/or 334 of the Fc region (EUnumbering of residues). In an exemplary embodiment, the antibodycomprising the following amino acid substitutions in its Fe region:S298A, E333A, and K334A.

In some embodiments, alterations are made in the Fc region that resultin altered (i.e., either improved or diminished) C1q binding and/orComplement Dependent Cytotoxicity (CDC), e.g., as described in U.S. Pat.No. 6,194,551, WO 99/51642, and Idusogie et al. J. Immunol. 164:4178-4184 (2000).

Antibodies with increased half lives and improved binding to theneonatal Fc receptor (FcRn), which is responsible for the transfer ofmaternal IgGs to the fetus (Guyer et al., J. Immunol. 117:587 (1976) andKim et al., J. Immunol. 24:249 (1994)), are described inUS2005/0014934A1 (Hinton et al.)). Those antibodies comprise an Fcregion with one or more substitutions therein which improve binding ofthe Fc region to FcRn. Such Fc variants include those with substitutionsat one or more of Fc region residues: 238, 256, 265, 272, 286, 303, 305,307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, 413, 424 or434, e.g., substitution of Fc region residue 434 (U.S. Pat. No.7,371,826). See also Duncan & Winter, Nature 322:738-40 (1988); U.S.Pat. Nos. 5,648,260; 5,624,821; and WO 94/29351 concerning otherexamples of Fc region variants.

(xii) Antibody Derivatives

The antibodies used in the invention can be further modified to containadditional nonproteinaceous moieties that are known in the art andreadily available. In certain embodiments, the moieties suitable forderivatization of the antibody are water soluble polymers. Non-limitingexamples of water soluble polymers include, but are not limited to,polyethylene glycol (PEG), copolymers of ethylene glycol/propyleneglycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene/maleicanhydride copolymer, polyaminoacids (either homopolymers or randomcopolymers), and dextran or poly(n-vinyl pyrrolidone)polyethyleneglycol, propropylene glycol homopolymers, prolypropylene oxide/ethyleneoxide co-polymers, polyoxyethylated polyols (e.g., glycerol), polyvinylalcohol, and mixtures thereof. Polyethylene glycol propionaldehyde mayhave advantages in manufacturing due to its stability in water. Thepolymer may be of any molecular weight, and may be branched orunbranched. The number of polymers attached to the antibody may vary,and if more than one polymer are attached, they can be the same ordifferent molecules. In general, the number and/or type of polymers usedfor derivatization can be determined based on considerations including,but not limited to, the particular properties or functions of theantibody to be improved, whether the antibody derivative will be used ina therapy under defined conditions, etc.

(xiii) Vectors, Host Cells, and Recombinant Methods

Antibodies may also be produced using recombinant methods. Forrecombinant production of an anti-antigen antibody, nucleic acidencoding the antibody is isolated and inserted into a replicable vectorfor further cloning (amplification of the DNA) or for expression. DNAencoding the antibody may be readily isolated and sequenced usingconventional procedures (e.g., by using oligonucleotide probes that arecapable of binding specifically to genes encoding the heavy and lightchains of the antibody). Many vectors are available. The vectorcomponents generally include, but are not limited to, one or more of thefollowing: a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence.

(a) Signal Sequence Component

An antibody may be produced recombinantly not only directly, but also asa fusion polypeptide with a heterologous polypeptide, which ispreferably a signal sequence or other polypeptide having a specificcleavage site at the N-terminus of the mature protein or polypeptide.The heterologous signal sequence selected preferably is one that isrecognized and processed (e.g., cleaved by a signal peptidase) by thehost cell. For prokaryotic host cells that do not recognize and processa native antibody signal sequence, the signal sequence is substituted bya prokaryotic signal sequence selected, for example, from the group ofthe alkaline phosphatase, penicillinase, 1pp, or heat-stable enterotoxinII leaders. For yeast secretion the native signal sequence may besubstituted by, e.g., the yeast invertase leader, a factor leader(including Saccharomyces and Kluyveromyces α-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex gD signal, are available.

(b) Origin of Replication

Both expression and cloning vectors contain a nucleic acid sequence thatenables the vector to replicate in one or more selected host cells.Generally, in cloning vectors this sequence is one that enables thevector to replicate independently of the host chromosomal DNA, andincludes origins of replication or autonomously replicating sequences.Such sequences are well known for a variety of bacteria, yeast, andviruses. The origin of replication from the plasmid pBR322 is suitablefor most Gram-negative bacteria, the 2μ, plasmid origin is suitable foryeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV)are useful for cloning vectors in mammalian cells. Generally, the originof replication component is not needed for mammalian expression vectors(the SV40 origin may typically be used only because it contains theearly promoter.

(c) Selection Gene Component

Expression and cloning vectors may contain a selection gene, also termeda selectable marker. Typical selection genes encode proteins that (a)confer resistance to antibiotics or other toxins, e.g., ampicillin,neomycin, methotrexate, or tetracycline, (b) complement auxotrophicdeficiencies, or (c) supply critical nutrients not available fromcomplex media, e.g., the gene encoding D-alanine racemase for Bacilli.

One example of a selection scheme utilizes a drug to arrest growth of ahost cell. Those cells that are successfully transformed with aheterologous gene produce a protein conferring drug resistance and thussurvive the selection regimen. Examples of such dominant selection usethe drugs neomycin, mycophenolic acid and hygromycin.

Another example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take upantibody-encoding nucleic acid, such as DHFR, glutamine synthetase (GS),thymidine kinase, metallothionein-I and —II, preferably primatemetallothionein genes, adenosine deaminase, ornithine decarboxylase,etc.

For example, cells transformed with the DHFR gene are identified byculturing the transformants in a culture medium containing methotrexate(Mtx), a competitive antagonist of DHFR. Under these conditions, theDHFR gene is amplified along with any other co-transformed nucleic acid.A Chinese hamster ovary (CHO) cell line deficient in endogenous DHFRactivity (e.g., ATCC CRL-9096) may be used.

Alternatively, cells transformed with the GS gene are identified byculturing the transformants in a culture medium containing L-methioninesulfoximine (Msx), an inhibitor of GS. Under these conditions, the GSgene is amplified along with any other co-transformed nucleic acid. TheGS selection/amplification system may be used in combination with theDHFR selection/amplification system described above.

Alternatively, host cells (particularly wild-type hosts that containendogenous DHFR) transformed or co-transformed with DNA sequencesencoding an antibody of interest, wild-type DHFR gene, and anotherselectable marker such as aminoglycoside 3′-phosphotransferase (APH) canbe selected by cell growth in medium containing a selection agent forthe selectable marker such as an aminoglycosidic antibiotic, e.g.,kanamycin, neomycin, or G418. See U.S. Pat. No. 4,965,199.

A suitable selection gene for use in yeast is the trp1 gene present inthe yeast plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979)). Thetrp1 gene provides a selection marker for a mutant strain of yeastlacking the ability to grow in tryptophan, for example, ATCC No. 44076or PEP4-1. Jones, Genetics, 85:12 (1977). The presence of the trp1lesion in the yeast host cell genome then provides an effectiveenvironment for detecting transformation by growth in the absence oftryptophan. Similarly, Leu2-deficient yeast strains (ATCC 20,622 or38,626) are complemented by known plasmids bearing the Leu2 gene.

In addition, vectors derived from the 1.6 μm circular plasmid pKD1 canbe used for transformation of Kluyveromyces yeasts. Alternatively, anexpression system for large-scale production of recombinant calfchymosin was reported for K. lactis. Van den Berg, Bio/Technology, 8:135(1990). Stable multi-copy expression vectors for secretion of maturerecombinant human serum albumin by industrial strains of Kluyveromyceshave also been disclosed. Fleer et al., Bio/Technology, 9:968-975(1991).

(d) Promoter Component

Expression and cloning vectors generally contain a promoter that isrecognized by the host organism and is operably linked to nucleic acidencoding an antibody. Promoters suitable for use with prokaryotic hostsinclude the phoA promoter, β-lactamase and lactose promoter systems,alkaline phosphatase promoter, a tryptophan (trp) promoter system, andhybrid promoters such as the tac promoter. However, other knownbacterial promoters are suitable. Promoters for use in bacterial systemsalso will contain a Shine-Dalgarno (S.D.) sequence operably linked tothe DNA encoding an antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryoticgenes have an AT-rich region located approximately 25 to 30 basesupstream from the site where transcription is initiated. Anothersequence found 70 to 80 bases upstream from the start of transcriptionof many genes is a CNCAAT region where N may be any nucleotide. At the3′ end of most eukaryotic genes is an AATAAA sequence that may be thesignal for addition of the poly A tail to the 3′ end of the codingsequence. All of these sequences are suitably inserted into eukaryoticexpression vectors.

Examples of suitable promoter sequences for use with yeast hosts includethe promoters for 3-phosphoglycerate kinase or other glycolytic enzymes,such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase,pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphateisomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphateisomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657. Yeast enhancers also are advantageously used with yeastpromoters.

Antibody transcription from vectors in mammalian host cells can becontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowlpox virus, adenovirus (such asAdenovirus 2), bovine papilloma virus, avian sarcoma virus,cytomegalovirus, a retrovirus, hepatitis-B virus, Simian Virus 40(SV40), or from heterologous mammalian promoters, e.g., the actinpromoter or an immunoglobulin promoter, from heat-shock promoters,provided such promoters are compatible with the host cell systems.

The early and late promoters of the SV40 virus are conveniently obtainedas an SV40 restriction fragment that also contains the SV40 viral originof replication. The immediate early promoter of the humancytomegalovirus is conveniently obtained as a HindIII E restrictionfragment. A system for expressing DNA in mammalian hosts using thebovine papilloma virus as a vector is disclosed in U.S. Pat. No.4,419,446. A modification of this system is described in U.S. Pat. No.4,601,978. See also Reyes et al., Nature 297:598-601 (1982) onexpression of human β-interferon cDNA in mouse cells under the controlof a thymidine kinase promoter from herpes simplex virus. Alternatively,the Rous Sarcoma Virus long terminal repeat can be used as the promoter.

(e) Enhancer Element Component

Transcription of a DNA encoding an antibody of this invention by highereukaryotes is often increased by inserting an enhancer sequence into thevector. Many enhancer sequences are now known from mammalian genes(globin, elastase, albumin, a-fetoprotein, and insulin). Typically,however, one will use an enhancer from a eukaryotic cell virus. Examplesinclude the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyomaenhancer on the late side of the replication origin, and adenovirusenhancers. See also Yaniv, Nature 297:17-18 (1982) on enhancing elementsfor activation of eukaryotic promoters. The enhancer may be spliced intothe vector at a position 5′ or 3′ to the antibody-encoding sequence, butis preferably located at a site 5′ from the promoter.

(f) Transcription Termination Component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human, or nucleated cells from other multicellularorganisms) will also contain sequences necessary for the termination oftranscription and for stabilizing the mRNA. Such sequences are commonlyavailable from the 5′ and, occasionally 3′, untranslated regions ofeukaryotic or viral DNAs or cDNAs. These regions contain nucleotidesegments transcribed as polyadenylated fragments in the untranslatedportion of the mRNA encoding antibody. One useful transcriptiontermination component is the bovine growth hormone polyadenylationregion. See WO94/11026 and the expression vector disclosed therein.

(g) Selection and Transformation of Host Cells

Suitable host cells for cloning or expressing the DNA in the vectorsherein are the prokaryote, yeast, or higher eukaryote cells describedabove. Suitable prokaryotes for this purpose include eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as Escherichia, e.g., E. coli, Enterobacter,Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium,Serratia, e.g., Serratia marcescans, and Shigella, as well as Bacillisuch as B. subtilis and B. licheniformis (e.g., B. licheniformis 41Pdisclosed in DD 266,710 published 12 Apr. 1989), Pseudomonas such as P.aeruginosa, and Streptomyces. One preferred E. coli cloning host is E.coli 294 (ATCC 31,446), although other strains such as E. coli B, E.coli X1776 (ATCC 31,537), and E. coli W3110 (ATCC 27,325) are suitable.These examples are illustrative rather than limiting.

Full length antibody, antibody fusion proteins, and antibody fragmentscan be produced in bacteria, in particular when glycosylation and Fceffector function are not needed, such as when the therapeutic antibodyis conjugated to a cytotoxic agent (e.g., a toxin) that by itself showseffectiveness in tumor cell destruction. Full length antibodies havegreater half-life in circulation. Production in E. coli is faster andmore cost efficient. For expression of antibody fragments andpolypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et.al.), U.S. Pat. No. 5,789,199 (Joly et al.), U.S. Pat. No. 5,840,523(Simmons et al.), which describes translation initiation region (TIR)and signal sequences for optimizing expression and secretion. See alsoCharlton, Methods in Molecular Biology, Vol. 248 (B. K. C. Lo, ed.,Humana Press, Totowa, N.J., 2003), pp. 245-254, describing expression ofantibody fragments in E. coli. After expression, the antibody may beisolated from the E. coli cell paste in a soluble fraction and can bepurified through, e.g., a protein A or G column depending on theisotype. Final purification can be carried out similar to the processfor purifying antibody expressed e.g., in CHO cells.

In addition to prokaryotes, eukaryotic microbes such as filamentousfungi or yeast are suitable cloning or expression hosts forantibody-encoding vectors. Saccharomyces cerevisiae, or common baker'syeast, is the most commonly used among lower eukaryotic hostmicroorganisms. However, a number of other genera, species, and strainsare commonly available and useful herein, such as Schizosaccharomycespombe; Kluyveromyces hosts such as, e.g., K. lactis, K. fragilis (ATCC12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K.waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans,and K. marxianus; yarrowia (EP 402,226); Pichia pastoris (EP 183,070);Candida; Trichoderma reesia (EP 244,234); Neurospora crassa;Schwanniomyces such as Schwanniomyces occidentalis; and filamentousfungi such as, e.g., Neurospora, Penicillium, Tolypocladium, andAspergillus hosts such as A. nidulans and A. niger. For a reviewdiscussing the use of yeasts and filamentous fungi for the production oftherapeutic proteins, see, e.g., Gerngross, Nat. Biotech. 22:1409-1414(2004).

Certain fungi and yeast strains may be selected in which glycosylationpathways have been “humanized,” resulting in the production of anantibody with a partially or fully human glycosylation pattern. See,e.g., Li et al., Nat. Biotech. 24:210-215 (2006) (describinghumanization of the glycosylation pathway in Pichia pastoris); andGerngross et al., supra.

Suitable host cells for the expression of glycosylated antibody are alsoderived from multicellular organisms (invertebrates and vertebrates).Examples of invertebrate cells include plant and insect cells. Numerousbaculoviral strains and variants and corresponding permissive insecthost cells from hosts such as Spodoptera frugiperda (caterpillar), Aedesaegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster(fruitfly), and Bombyx mori have been identified. A variety of viralstrains for transfection are publicly available, e.g., the L-1 variantof Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV,and such viruses may be used as the virus herein according to theinvention, particularly for transfection of Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato,duckweed (Leninaceae), alfalfa (M. truncatula), and tobacco can also beutilized as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498,6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIES™ technologyfor producing antibodies in transgenic plants).

Vertebrate cells may be used as hosts, and propagation of vertebratecells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines are monkey kidney CV1 linetransformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line(293 or 293 cells subcloned for growth in suspension culture, Graham etal., J. Gen Virol. 36:59 (1977)); baby hamster kidney cells (BHK, ATCCCCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod. 23:243-251(1980)); monkey kidney cells (CV1 ATCC CCL 70); African green monkeykidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells(HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo ratliver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci.383:44-68 (1982)); MRC 5 cells; FS4 cells; and a human hepatoma line(Hep G2). Other useful mammalian host cell lines include Chinese hamsterovary (CHO) cells, including DHFR⁻ CHO cells (Urlaub et al., Proc. Natl.Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 andSp2/0. For a review of certain mammalian host cell lines suitable forantibody production, see, e.g., Yazaki and Wu, Methods in MolecularBiology, Vol. 248 (B. K. C. Lo, ed., Humana Press, Totowa, N.J., 2003),pp. 255-268.

Host cells are transformed with the above-described expression orcloning vectors for antibody production and cultured in conventionalnutrient media modified as appropriate for inducing promoters, selectingtransformants, or amplifying the genes encoding the desired sequences.

(h) Culturing the Host Cells

The host cells used to produce an antibody of this invention may becultured in a variety of media. Commercially available media such asHam's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640(Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) aresuitable for culturing the host cells. In addition, any of the mediadescribed in Ham et al., Meth. Enz. 58:44 (1979), Barnes et al., Anal.Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866; 4,927,762;4,560,655; or 5,122,469; WO 90/03430; WO 87/00195; or U.S. Pat. Re.30,985 may be used as culture media for the host cells. Any of thesemedia may be supplemented as necessary with hormones and/or other growthfactors (such as insulin, transferrin, or epidermal growth factor),salts (such as sodium chloride, calcium, magnesium, and phosphate),buffers (such as HEPES), nucleotides (such as adenosine and thymidine),antibiotics (such as GENTAMYCIN™ drug), trace elements (defined asinorganic compounds usually present at final concentrations in themicromolar range), and glucose or an equivalent energy source. Any othernecessary supplements may also be included at appropriate concentrationsthat would be known to those skilled in the art. The culture conditions,such as temperature, pH, and the like, are those previously used withthe host cell selected for expression, and will be apparent to theordinarily skilled artisan.

(xiv) Purification of Antibody

When using recombinant techniques, the antibody can be producedintracellularly, in the periplasmic space, or directly secreted into themedium. If the antibody is produced intracellularly, as a first step,the particulate debris, either host cells or lysed fragments, areremoved, for example, by centrifugation or ultrafiltration. Carter etal., Bio/Technology 10:163-167 (1992) describe a procedure for isolatingantibodies which are secreted to the periplasmic space of E. coli.Briefly, cell paste is thawed in the presence of sodium acetate (pH3.5), EDTA, and phenylmethylsulfonylfluoride (PMSF) over about 30 min.Cell debris can be removed by centrifugation. Where the antibody issecreted into the medium, supernatants from such expression systems aregenerally first concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

The antibody composition prepared from the cells can be purified using,for example, hydroxylapatite chromatography, hydrophobic interactionchromatography, gel electrophoresis, dialysis, and affinitychromatography, with affinity chromatography being among one of thetypically preferred purification steps. The suitability of protein A asan affinity ligand depends on the species and isotype of anyimmunoglobulin Fc domain that is present in the antibody. Protein A canbe used to purify antibodies that are based on human γ1, γ2, or γ4 heavychains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)). Protein G isrecommended for all mouse isotypes and for human γ3 (Guss et al., EMBOJ. 5:15671575 (1986)). The matrix to which the affinity ligand isattached is most often agarose, but other matrices are available.Mechanically stable matrices such as controlled pore glass orpoly(styrenedivinyl)benzene allow for faster flow rates and shorterprocessing times than can be achieved with agarose. Where the antibodycomprises a C_(H)3 domain, the Bakerbond ABX™ resin (J. T. Baker,Phillipsburg, N.J.) is useful for purification. Other techniques forprotein purification such as fractionation on an ion-exchange column,ethanol precipitation, Reverse Phase HPLC, chromatography on silica,chromatography on heparin SEPHAROSE™ chromatography on an anion orcation exchange resin (such as a polyaspartic acid column),chromatofocusing, SDS-PAGE, and ammonium sulfate precipitation are alsoavailable depending on the antibody to be recovered.

In general, various methodologies for preparing antibodies for use inresearch, testing, and clinical are well-established in the art,consistent with the above-described methodologies and/or as deemedappropriate by one skilled in the art for a particular antibody ofinterest.

C. Selecting Biologically Active Antibodies

Antibodies produced as described above may be subjected to one or more“biological activity” assays to select an antibody with beneficialproperties from a therapeutic perspective or selecting formulations andconditions that retain biological activity of the antibody. The antibodymay be tested for its ability to bind the antigen against which it wasraised. For example, methods known in the art (such as ELISA, WesternBlot, etc.) may be used.

For example, for an anti-PD-L1 antibody, the antigen binding propertiesof the antibody can be evaluated in an assay that detects the ability tobind to PD-L1. In some embodiments, the binding of the antibody may bedetermined by saturation binding; ELISA; and/or competition assays (e.g.RIA's), for example. Also, the antibody may be subjected to otherbiological activity assays, e.g., in order to evaluate its effectivenessas a therapeutic. Such assays are known in the art and depend on thetarget antigen and intended use for the antibody. For example, thebiological effects of PD-L1 blockade by the antibody can be assessed inCD8+ T cells, a lymphocytic choriomeningitis virus (LCMV) mouse modeland/or a syngeneic tumor model e.g., as described in U.S. Pat. No.8,217,149.

To screen for antibodies which bind to a particular epitope on theantigen of interest (e.g., those which block binding of the anti-PD-L1antibody of the example to PD-L1), a routine cross-blocking assay suchas that described in Antibodies, A Laboratory Manual, Cold Spring HarborLaboratory, Ed Harlow and David Lane (1988), can be performed.Alternatively, epitope mapping, e.g. as described in Champe et al., J.Biol. Chem. 270:1388-1394 (1995), can be performed to determine whetherthe antibody binds an epitope of interest.

D. Pharmaceutical Compositions and Formulations

Also provided herein are pharmaceutical compositions and formulationscomprising a PD-1 axis binding antagonist and/or an antibody describedherein (such as an anti-PD-L1 antibody, or a bispecific antibody thatbinds CEA and CD3) and a pharmaceutically acceptable carrier.

Pharmaceutical compositions and formulations as described herein can beprepared by mixing the active ingredients (e.g. a PD-1 axis bindingantagonist and/or an anti-CEA/anti-CD3 bispecific antibody) having thedesired degree of purity with one or more optional pharmaceuticallyacceptable carriers (Remington's Pharmaceutical Sciences 16th edition,Osol, A. Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Pharmaceutically acceptable carriers are generally nontoxicto recipients at the dosages and concentrations employed, and include,but are not limited to: buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride; benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as polyethylene glycol(PEG). Exemplary pharmaceutically acceptable carriers herein furtherinclude insterstitial drug dispersion agents such as solubleneutral-active hyaluronidase glycoproteins (sHASEGP), for example, humansoluble PH-20 hyaluronidase glycoproteins, such as rHuPH20 (HYLENEX®,Baxter International, Inc.). Certain exemplary sHASEGPs and methods ofuse, including rHuPH20, are described in US Patent Publication Nos.2005/0260186 and 2006/0104968. In one aspect, a sHASEGP is combined withone or more additional glycosaminoglycanases such as chondroitinases.

Exemplary lyophilized antibody formulations are described in U.S. Pat.No. 6,267,958. Aqueous antibody formulations include those described inU.S. Pat. No. 6,171,586 and WO2006/044908, the latter formulationsincluding a histidine-acetate buffer.

The composition and formulation herein may also contain more than oneactive ingredients as necessary for the particular indication beingtreated, preferably those with complementary activities that do notadversely affect each other. Such active ingredients are suitablypresent in combination in amounts that are effective for the purposeintended.

Active ingredients may be entrapped in microcapsules prepared, forexample, by coacervation techniques or by interfacial polymerization,for example, hydroxymethylcellulose or gelatin-microcapsules andpoly-(methylmethacylate) microcapsules, respectively, in colloidal drugdelivery systems (for example, liposomes, albumin microspheres,microemulsions, nano-particles and nanocapsules) or in macroemulsions.Such techniques are disclosed in Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. (1980).

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. The formulationsto be used for in vivo administration are generally sterile. Sterilitymay be readily accomplished, e.g., by filtration through sterilefiltration membranes.

IV. Methods of Treatment

Provided herein are methods for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody. In some embodiments, thetreatment results in a response in the individual after treatment. Insome embodiments, the response is a partial response. In someembodiments, the response is a complete response. In some embodiments,the treatment results in a sustained response (e.g., a sustained partialresponse or complete response) in the individual after cessation of thetreatment. The methods described herein may find use in treatingconditions where enhanced immunogenicity is desired such as increasingtumor immunogenicity for the treatment of cancer. Also provided hereinare methods of enhancing immune function in an individual having cancercomprising administering to the individual an effective amount of a PD-1axis binding antagonist (e.g., MPDL3280A) and an anti-CEA/anti-CD3bispecific antibody (e.g. CEA TCB).

In some instances, the methods provided herein include administration ofan effective amount of a PD-1 axis binding antagonist selected from thegroup consisting of a PD-1 binding antagonist, a PD-L1 bindingantagonist, and a PD-L2 binding antagonist. In some instances, the PD-L1binding antagonist is an antibody, such as an antibody that is capableof inhibiting PD-L1 binding to PD-1 and B7.1, but does not disruptbinding of PD-1 to PD-L2. In some instances, the PD-L1 bindingantagonist antibody is MPDL3280A, which may be administered at a dose ofabout 800 mg to about 1500 mg every three weeks (e.g., about 1000 mg toabout 1300 mg every three weeks, e.g., about 1100 mg to about 1200 mgevery three weeks). In some embodiments, MPDL3280A is administered at adose of about 1200 mg every three weeks.

As a general proposition, the therapeutically effective amount of a PD-1axis binding antagonist (e.g., an anti-PD-L1 antibody, e.g., MPDL3280A)may be administered to a human will be in the range of about 0.01 toabout 50 mg/kg of patient body weight whether by one or moreadministrations. In some embodiments, for example, the antagonist (e.g.,anti-PD-L1 antibody, e.g., MPDL3280A) is administered in a dose of about0.01 to about 45 mg/kg, about 0.01 to about 40 mg/kg, about 0.01 toabout 35 mg/kg, about 0.01 to about 30 mg/kg, about 0.01 to about 25mg/kg, about 0.01 to about 20 mg/kg, about 0.01 to about 15 mg/kg, about0.01 to about 10 mg/kg, about 0.01 to about 5 mg/kg, or about 0.01 toabout 1 mg/kg administered daily, for example. In some embodiments, theantagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administeredat 15 mg/kg. However, other dosage regimens may be useful. In oneembodiment, a PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody,e.g., MPDL3280A) is administered to a human at a dose of about 100 mg,about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg,about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg,about 1200 mg, about 1300 mg, about 1400 mg, or about 1500 mg. In someembodiments, a PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody,e.g., MPDL3280A) is administered at a dose of about 1150 mg to about1250 mg every three weeks. In some embodiments, a PD-1 axis bindingantagonist (e.g., anti-PD-L1 antibody, e.g., MPDL3280A) is administeredat a dose of about 1200 mg every three weeks. The dose may beadministered as a single dose or as multiple doses (e.g., 2 or 3 doses),such as infusions. The dose of the antibody administered in acombination treatment may be reduced as compared to a single treatment.In some embodiments, for example, the method for treating or delayingprogression of cancer in an individual comprises a dosing regimencomprising treatment cycles, wherein the individual is administered, ondays 1 of each cycle, a human PD-1 axis binding antagonist (e.g.,anti-PD-L1 antibody, e.g., MPDL3280A) at a dose of about 1200 mg,wherein each cycle is 21 days (i.e., each cycle is repeated every 21days). The progress of this therapy is easily monitored by conventionaltechniques.

In some instances, the methods provided herein include administration ofan effective amount of an anti-CEA/anti-CD3 bispecific antibody (e.g.,CEA TCB). In some instances, the anti-CEA/anti-CD3 bispecific antibody(e.g., CEA TCB) is administered to the individual at a dose of about 5mg to about 400 mg every week (e.g., about 10 mg to about 60 mg everyweek, e.g., about 10 mg to about 40 mg every week, e.g. about 80 mg toabout 200 mg every week, e.g. about 80 mg to about 400 mg every week, ore.g. about 160 mg to 400 mg every week). In some embodiments, theanti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB) is administered ata dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg, about 80 mgor about 160 mg, every week. As a general proposition, thetherapeutically effective amount of an anti-CEA/anti-CD3 bispecificantibody (e.g., CEA TCB) administered to a human will be in the range ofabout 5 to about 400 mg (e.g., about 5 mg, about 10 mg, about 15 mg,about 20 mg, about 25 mg, about 30 mg about 35 mg, about 40 mg, about 45mg, about 50 mg, about 55 mg, about 60 mg, about 65 mg, about 70 mg,about 75 mg, about 80 mg about 85 mg, about 90 mg, about 95 mg, about100 mg, about 150 mg, about 160 mg, about 200 mg, about 250 mg, about300 mg, about 350 mg, or about 400 mg), whether by one or moreadministrations. For example, in some embodiments, about 5 mg, about 10mg, about 20 mg, about 40 mg, about 80 mg or about 160 mg, of CEA TCB isadministered. In some embodiments, CEA TCB is administered at 5 mg, 10mg, 20 mg, 40 mg, 80 mg or 160 mg, once a week. In particularembodiments, at least about 80 mg of CEA TCB is administered. In someembodiments, CEA TCB is administered at 80 mg once a week. In someembodiments, CEA TCB is administered at 80-200 mg, 80-400 mg or 160-400mg once a week. In some embodiments, the anti-CEA/anti-CD3 bispecificantibody (e.g., CEA TCB) may be administered weekly, every 2 weeks,every 3 weeks, every 4 weeks, on days 1, 8 and 15 of each 21-day cycle,or on days 1, 8, and 15 of each 28-day cycle.

In some instances, the PD-1 axis binding antagonist (e.g., an anti-PD-L1antibody, e.g., MPDL3280A) and the anti-CEA/anti-CD3 bispecific antibody(e.g., CEA TCB) are administered in a single dosing regimen. Theadministration of these agents may be concurrent or separate within thecontext of the dosing regimen. For example, in some instances, themethods provided herein include a dosing regimen comprising treatmentcycles, wherein the individual is administered, on days 1 of each cycle,a human PD-1 axis binding antagonist at a dose of about 1200 mg, and ondays 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecificantibody at a dose of about 5 mg, about 10 mg, about 20 mg, about 40 mg,about 80 mg or about 160 mg, each cycle being repeated every 21 days. Inone embodiment, the individual is administered, on days 1 of each cycle,a human PD-1 axis binding antagonist at a dose of about 1200 mg, and ondays 1, 8, and 15 of each cycle, an anti-CEA/anti-CD3 bispecificantibody at a dose of about 80 mg, each cycle being repeated every 21days. In a particular embodiment, the individual is administered, ondays 1 of each cycle, a human PD-1 axis binding antagonist at a dose ofabout 1200 mg, and on days 1, 8, and 15 of each cycle, ananti-CEA/anti-CD3 bispecific antibody at a dose of at least about 80 mg,each cycle being repeated every 21 days. In still a further embodiment,the individual is administered, on days 1 of each cycle, a human PD-1axis binding antagonist at a dose of about 1200 mg, and on days 1, 8,and 15 of each cycle, an anti-CEA/anti-CD3 bispecific antibody at a doseof about 80 mg to about 200 mg. about 80 mg to about 400 mg, or about160 mg to about 400 mg, each cycle being repeated every 21 days.

In some embodiments, the individual is a human. In some embodiments, theindividual is suffering from locally advanced or metastatic breastcancer. In some embodiments, the individual has CEA positive cancer. Insome embodiments, CEA positive cancer is colon cancer, lung cancer,ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer,endometrial cancer, breast cancer, kidney cancer, esophageal cancer, orprostate cancer. In a particular embodiment, the cancer is colorectalcarcinoma. In some embodiments, the breast cancer is a breast carcinomaor a breast adenocarcinoma. In some embodiments, the breast carcinoma isan invasive ductal carcinoma. In some embodiments, the lung cancer is alung adenocarcinoma. In some embodiments, the colon cancer is acolorectal adenocarcinoma. In some embodiments, the cancer cells in theindividual express PD-L1. In some embodiments, the cancer cells in theindividual express CEA protein at a level that is detectable (e.g.,detectable using methods known in the art).

In some embodiments, the individual has been treated with a cancertherapy before the combination treatment with a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody. In someembodiments, the individual has cancer that is resistant to one or morecancer therapies. In some embodiments, resistance to cancer therapyincludes recurrence of cancer or refractory cancer. Recurrence may referto the reappearance of cancer, in the original site or a new site, aftertreatment. In some embodiments, resistance to a cancer therapy includesprogression of the cancer during treatment with the anti-cancer therapy.In some embodiments, resistance to a cancer therapy includes cancer thatdoes not response to treatment. The cancer may be resistant at thebeginning of treatment or it may become resistant during treatment. Insome embodiments, the cancer is at early stage or at late stage.

In some embodiments, the combination therapy of the invention comprisesadministration of a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody. The PD-1 axis binding antagonistand the anti-CEA/anti-CD3 bispecific antibody may be administered in anysuitable manner known in the art. For example, The PD-1 axis bindingantagonist and the anti-CEA/anti-CD3 bispecific antibody may beadministered sequentially (at different times) or concurrently (at thesame time). In some embodiments, the PD-1 axis binding antagonist is ina separate composition as the anti-CEA/anti-CD3 bispecific antibody. Insome embodiments, the PD-1 axis binding antagonist is in the samecomposition as the anti-CEA/anti-CD3 bispecific antibody.

The PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecificantibody may be administered by the same route of administration or bydifferent routes of administration. In some embodiments, the PD-1 axisbinding antagonist is administered intravenously, intramuscularly,subcutaneously, topically, orally, transdermally, intraperitoneally,intraorbitally, by implantation, by inhalation, intrathecally,intraventricularly, or intranasally. In some embodiments, theanti-CEA/anti-CD3 bispecific antibody is administered intravenously,intramuscularly, subcutaneously, topically, orally, transdermally,intraperitoneally, intraorbitally, by implantation, by inhalation,intrathecally, intraventricularly, or intranasally. An effective amountof the PD-1 axis binding antagonist and the anti-CEA/anti-CD3 bispecificantibody may be administered for prevention or treatment of disease. Theappropriate dosage of the PD-1 axis binding antagonist and/or theanti-CEA/anti-CD3 bispecific antibody may be determined based on thetype of disease to be treated, the type of the PD-1 axis bindingantagonist and the anti-CEA/anti-CD3 bispecific antibody, the severityand course of the disease, the clinical condition of the individual, theindividual's clinical history and response to the treatment, and thediscretion of the attending physician.

In some embodiments, the methods may further comprise an additionaltherapy. The additional therapy may be radiation therapy, surgery (e.g.,lumpectomy and a mastectomy), chemotherapy, gene therapy, DNA therapy,viral therapy, RNA therapy, immunotherapy, bone marrow transplantation,nanotherapy, monoclonal antibody therapy, or a combination of theforegoing. The additional therapy may be in the form of adjuvant orneoadjuvant therapy. In some embodiments, the additional therapy is theadministration of small molecule enzymatic inhibitor or anti-metastaticagent. In some embodiments, the additional therapy is the administrationof side-effect limiting agents (e.g., agents intended to lessen theoccurrence and/or severity of side effects of treatment, such asanti-nausea agents, etc.). In some embodiments, the additional therapyis radiation therapy. In some embodiments, the additional therapy issurgery. In some embodiments, the additional therapy is a combination ofradiation therapy and surgery. In some embodiments, the additionaltherapy is gamma irradiation. In some embodiments, the additionaltherapy is therapy targeting PI3K/AKT/mTOR pathway, HSP90 inhibitor,tubulin inhibitor, apoptosis inhibitor, and/or chemopreventative agent.The additional therapy may be one or more of the chemotherapeutic agentsdescribed herein.

Other Combination Therapies

Also provided herein are methods for treating or delaying progression ofcancer in an individual comprising administering to the individual ahuman PD-1 axis binding antagonist (e.g., anti-PD-L1 antibody, e.g.,MPDL3280A) and an anti-CEA/anti-CD3 bispecific antibody (e.g., CEA TCB)in conjunction with another anti-cancer agent or cancer therapy.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith a chemotherapy or chemotherapeutic agent. In some embodiments, aPD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody may be administered in conjunction with a radiation therapy orradiotherapeutic agent. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with a targeted therapy or targetedtherapeutic agent. In some embodiments, a PD-1 axis binding antagonistand an anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with an immunotherapy or immunotherapeutic agent, forexample a monoclonal antibody.

Without wishing to be bound to theory, it is thought that enhancing Tcell stimulation, by promoting an activating co-stimulatory molecule orby inhibiting a negative co-stimulatory molecule, may promote tumor celldeath thereby treating or delaying progression of cancer. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with an agonistdirected against an activating co-stimulatory molecule. In someembodiments, an activating co-stimulatory molecule may include CD40,CD226, CD28, OX40, GITR, CD137, CD27, HVEM, or CD127. In someembodiments, the agonist directed against an activating co-stimulatorymolecule is an agonist antibody that binds to CD40, CD226, CD28, OX40,GITR, CD137, CD27, HVEM, or CD127. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an antagonist directed against aninhibitory co-stimulatory molecule. In some embodiments, an inhibitoryco-stimulatory molecule may include CTLA-4 (also known as CD152), PD-1,TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT, MICA/B, orarginase. In some embodiments, the antagonist directed against aninhibitory co-stimulatory molecule is an antagonist antibody that bindsto CTLA-4, PD-1, TIM-3, BTLA, VISTA, LAG-3, B7-H3, B7-H4, IDO, TIGIT,MICA/B, or arginase.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antagonist directed against CTLA-4 (also known as CD152), e.g.,a blocking antibody. In some embodiments, a PD-1 axis binding antagonistmay be administered in conjunction with ipilimumab (also known asMDX-010, MDX-101, or YERVOY®). In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with tremelimumab (also known as ticilimumabor CP-675,206). In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with an antagonist directed against B7-H3 (also known asCD276), e.g., a blocking antibody. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with MGA271. In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with an antagonist directed against a TGFbeta, e.g., metelimumab (also known as CAT-192), fresolimumab (alsoknown as GC1008), or LY2157299.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith a treatment comprising adoptive transfer of a T cell (e.g., acytotoxic T cell or CTL) expressing a chimeric antigen receptor (CAR).In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith a treatment comprising adoptive transfer of a T cell comprising adominant-negative TGF beta receptor, e.g, a dominant-negative TGF betatype II receptor. In some embodiments, a PD-1 axis binding antagonistand an anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with a treatment comprising a HERCREEM protocol (see, e.g.,ClinicalTrials.gov Identifier NCT00889954).

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an agonist directed against CD137 (also known as TNFRSF9, 4-1BB, orILA), e.g., an activating antibody. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with urelumab (also known as BMS-663513). Insome embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an agonist directed against CD40, e.g., an activating antibody. Insome embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith CP-870893. In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with an agonist directed against OX40 (also known as CD134),e.g., an activating antibody. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an anti-OX40 antibody (e.g., AgonOX).In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an agonist directed against CD27, e.g., an activating antibody. Insome embodiments, a PD-1 axis binding antagonist may be administered inconjunction with CDX-1127. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an antagonist directed againstindoleamine-2,3-dioxygenase (IDO). In some embodiments, with the IDOantagonist is 1-methyl-D-tryptophan (also known as 1-D-MT).

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antibody-drug conjugate. In some embodiments, the antibody-drugconjugate comprises mertansine or monomethyl auristatin E (MMAE). Insome embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith and anti-NaPi2b antibody-MMAE conjugate (also known as DNIB0600A orRG7599). In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith trastuzumab emtansine (also known as T-DM1, ado-trastuzumabemtansine, or KADCYLA®, Genentech). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with DMUC5754A. In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with an antibody-drug conjugate targetingthe endothelin B receptor (EDNBR), e.g., an antibody directed againstEDNBR conjugated with MMAE.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an angiogenesis inhibitor. In some embodiments, a PD-1 axis bindingantagonist may be administered in conjunction with an antibody directedagainst a VEGF, e.g., VEGF-A. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with bevacizumab (also known as AVASTIN®,Genentech). In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antibody directed against angiopoietin 2 (also known as Ang2).In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith MEDI3617.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antineoplastic agent. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an agent targeting CSF-1R (also knownas M-CSFR or CD115). In some embodiments, a PD-1 axis binding antagonistand an anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with anti-CSF-1R (also known as IMC-CS4). In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with aninterferon, for example interferon alpha or interferon gamma. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with Roferon-A(also known as recombinant Interferon alpha-2a). In some embodiments, aPD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody may be administered in conjunction with GM-CSF (also known asrecombinant human granulocyte macrophage colony stimulating factor, rhuGM-CSF, sargramostim, or Leukine®). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with IL-2 (also known as aldesleukin orProleukin®). In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith IL-12. In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antibody targeting CD20. In some embodiments, the antibodytargeting CD20 is obinutuzumab (also known as GA101 or Gazyva®) orrituximab. In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an antibody targeting GITR. In some embodiments, the antibodytargeting GITR is TRX518.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith a cancer vaccine. In some embodiments, the cancer vaccine is apeptide cancer vaccine, which in some embodiments is a personalizedpeptide vaccine. In some embodiments the peptide cancer vaccine is amultivalent long peptide, a multi-peptide, a peptide cocktail, a hybridpeptide, or a peptide-pulsed dendritic cell vaccine (see, e.g., Yamadaet al., Cancer Sci, 104:14-21, 2013). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an adjuvant. In some embodiments, aPD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody may be administered in conjunction with a treatment comprisinga TLR agonist, e.g., Poly-ICLC (also known as Hiltonol®), LPS, MPL, orCpG ODN. In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith tumor necrosis factor (TNF) alpha. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with IL-1. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with HMGB1. In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an IL-10 antagonist. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with an IL-4antagonist. In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an IL-13 antagonist. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an HVEM antagonist. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with an ICOSagonist, e.g., by administration of ICOS-L, or an agonistic antibodydirected against ICOS. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with a treatment targeting CX3CL1. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with a treatmenttargeting CXCL9. In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with a treatment targeting CXCL10. In some embodiments, aPD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody may be administered in conjunction with a treatment targetingCCL5. In some embodiments, a PD-1 axis binding antagonist may beadministered in conjunction with an LFA-1 or ICAM1 agonist. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with a Selectinagonist.

In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith a targeted therapy. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an inhibitor of B-Raf. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with vemurafenib(also known as Zelboraf®). In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with dabrafenib (also known as Tafinlar®).In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith erlotinib (also known as Tarceva®). In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with an inhibitor of a MEK, such as MEK1(also known as MAP2K1) or MEK2 (also known as MAP2K2). In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with cobimetinib(also known as GDC-0973 or XL-518). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with trametinib (also known as Mekinist®).In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith an inhibitor of K-Ras. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an inhibitor of c-Met. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with onartuzumab(also known as MetMAb). In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an inhibitor of Alk. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with AF802 (alsoknown as CH5424802 or alectinib). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an inhibitor of a phosphatidylinositol3-kinase (PI3K). In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with BKM120. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with idelalisib (also known as GS-1101 orCAL-101). In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith perifosine (also known as KRX-0401). In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with an inhibitor of an Akt. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with MK2206. Insome embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith GSK690693. In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with GDC-0941. In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with an inhibitor of mTOR. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with sirolimus(also known as rapamycin). In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with temsirolimus (also known as CCI-779 orTorisel®). In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith everolimus (also known as RAD001). In some embodiments, a PD-1 axisbinding antagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with ridaforolimus (also known as AP-23573,MK-8669, or deforolimus). In some embodiments, a PD-1 axis bindingantagonist and an anti-CEA/anti-CD3 bispecific antibody may beadministered in conjunction with OSI-027. In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with AZD8055. In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with INK128. In some embodiments, a PD-1axis binding antagonist and an anti-CEA/anti-CD3 bispecific antibody maybe administered in conjunction with a dual PI3K/mTOR inhibitor. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with XL765. Insome embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith GDC-0980. In some embodiments, a PD-1 axis binding antagonist andan anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with BEZ235 (also known as NVP-BEZ235). In some embodiments,a PD-1 axis binding antagonist and an anti-CEA/anti-CD3 bispecificantibody may be administered in conjunction with BGT226. In someembodiments, a PD-1 axis binding antagonist and an anti-CEA/anti-CD3bispecific antibody may be administered in conjunction with GSK2126458.In some embodiments, a PD-1 axis binding antagonist and ananti-CEA/anti-CD3 bispecific antibody may be administered in conjunctionwith PF-04691502. In some embodiments, a PD-1 axis binding antagonistand an anti-CEA/anti-CD3 bispecific antibody may be administered inconjunction with PF-05212384 (also known as PKI-587).

V. Articles of Manufacture or Kits

In another embodiment of the invention, an article of manufacture or akit is provided comprising a PD-1 axis binding antagonist and/or ananti-CEA/anti-CD3 bispecific antibody. In some embodiments, the articleof manufacture or kit further comprises package insert comprisinginstructions for suing the PD-1 axis binding antagonist in conjunctionwith an anti-CEA/anti-CD3 bispecific antibody to treat or delayprogression of cancer in an individual or to enhance immune function ofan individual having cancer. Any of the PD-1 axis binding antagonistand/or an anti-CEA/anti-CD3 bispecific antibodies described herein maybe included in the article of manufacture or kits.

In some embodiments, the PD-1 axis binding antagonist and theanti-CEA/anti-CD3 bispecific antibody are in the same container orseparate containers. Suitable containers include, for example, bottles,vials, bags and syringes. The container may be formed from a variety ofmaterials such as glass, plastic (such as polyvinyl chloride orpolyolefin), or metal alloy (such as stainless steel or hastelloy). Insome embodiments, the container holds the formulation and the label on,or associated with, the container may indicate directions for use. Thearticle of manufacture or kit may further include other materialsdesirable from a commercial and user standpoint, including otherbuffers, diluents, filters, needles, syringes, and package inserts withinstructions for use. In some embodiments, the article of manufacturefurther includes one or more of another agent (e.g., a chemotherapeuticagent, and anti-neoplastic agent). Suitable containers for the one ormore agent include, for example, bottles, vials, bags and syringes.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

EXAMPLES

The invention will be more fully understood by reference to thefollowing examples. They should not, however, be construed as limitingthe scope of the invention. It is understood that the examples andembodiments described herein are for illustrative purposes only and thatvarious modifications or changes in light thereof will be suggested topersons skilled in the art and are to be included within the spirit andpurview of this application and scope of the appended claims.

Example 1: CEA TCB-Mediated Tumor Lysis Leads to Up-Regulation of PD-1on Human T Cells and of PD-L1 on Surviving Tumor Cells In Vitro

CEA TCB is a novel T cell bispecific antibody targeting thecarcinoembryonic antigen (CEA) on tumor cells and CD3 on T cells. It iscurrently being investigated as a single agent in a Phase I study inpatients with advanced and/or metastatic CEA expressing tumors.

The lysis of CEA-expressing MKN-45 target cells mediated by CEA TCB wasassessed after 24 h and 48 h of incubation with human PBMCs (E:T 10:1,LDH release) (FIG. 1A 24 h, FIG. 1B 48 h).

PBMCs were isolated from a buffy coat obtained from the “BlutspendeZurich”. Blood was diluted 3:1 with PBS. About 30 ml of the blood/PBSmixture was layered on 15 ml of Histopaque and centrifuged for 30 min at450 g without brake at room temperature (RT). Lymphocytes were collectedwith a 10 ml pipette into 50 ml tubes containing PBS and filled up to 50ml with PBS and centrifuged 10 min at 350 g. The supernatant wasdiscarded, the pellet re-suspended in 50 ml PBS and centrifuged for 10min at 300 g. The washing step was repeated once. Cells werere-suspended in RPMI containing 10% FCS and 1% GlutaMax and stored at37° C., 5% CO₂ in the incubator until the initiation of the assay.

Adherent MKN-45 target cells were harvested with Trypsin/EDTA one daybefore the assay, washed and plated at density of 1.4 mio cells/wellusing flat-bottom 24-well plates. Cells were left to adhere overnight ina humidified incubator. On the day of the assay, plates were centrifugedat 350 g for 5 min and the medium was aspirated. 550 μl per well ofassay medium (RPMI1640, 2% FCS, 1% GlutaMax) were added.

CEA TCB or untargeted TCB were added at indicated concentrations (rangeof 6.4 pM-100 nM, in triplicates). PBMCs were added to target cells atthe final Effector:Target ratio E:T 10:1, final volume per well was 1.1ml.

Target cell killing was assessed after 24 h and 48 h of incubation byquantification of LDH (lactate dehydrogenase) released into cellsupernatants by apoptotic/necrotic cells (LDH detection kit, RocheApplied Science, #11 644 793 001). Maximal lysis of the target cells(=100%) was achieved by incubation of target cells with 1% Triton X-100.Minimal lysis (=0%) refers to target cells co-incubated with effectorcells without bispecific antibody. The EC50 values were calculated usingGraphPadPrism5.

Following killing, the surface expression of PD-1 receptor (on CD4+ orCD8+ T cells) and of PD-L1 (on tumor cells that survived killing) wasassessed by flow cytometry.

Upon removal of 50 μl of the supernatant for the second LDH read-out(48h time point), the assay plates were centrifuged for 5 min at 350 gand the supernatants were discarded. Cell pellets were re-suspended in100 μl PBS and transferred into wells of a 96-round-bottom well plate.The wells were washed with 80 μl PBS and this washing step was pooledwith the already transferred supernatants from the steps before. Theadherent MKN45 cells were detached with 80 μl Cell Dissociation Buffer(Gibco) and transferred to the FACS plate as well. Plates werecentrifuged for 4 min at 400 g. Cell pellets were washed once again with150 μl of FACS-buffer. After centrifugation (400 g for 4 min), thesupernatant was removed and cells were re-suspended by carefulvortexing. The cell suspension per well was split in two and the assayplates were centrifuged again. 25 μl of the antibody mix (antibodiesdirected against CD4, CD8, PD-1 (Biolegend, #329920) and PD-L1(Biolegend #329708)) were added per well. To remove unbound antibody,the cells were washed twice with 150 μl FACS buffer per well. Aftercentrifugation (400 g for 4 min), the supernatant was removed and cellswere re-suspended in 120 μl FACS buffer.

FIG. 2 displays a dose-dependent up-regulation of PD-1 receptor onCD8+(A) and on CD4+(B) T cells as well as of PD-L1 on tumor cells thatresisted to killing (C) and were harvested after 48 h of incubation.

Example 2: CEA TCB-Mediated Tumor Lysis Leads to Up-Regulation of PD-1on Human T Cells and of PD-L1 on Surviving Tumor Cells In Vivo

Nonclinical in vivo pharmacology studies were conducted using a humancolon carcinoma xenograft tumor model (LS174T-fluc2) in immunodeficientNOG mice. Two different experimental settings were used: 1) tumor cellswere co-grafted with human PBMCs (subcutaneous [SC]) at different E:Tratios, and 2) tumor cells were injected SC followed by intraperitoneal(IP) transfer of PBMCs once tumors were established (reached a palpablemass).

Xenograft Experiment with Co-Grafting of Effector Cells

Results of CEA TCB in vivo efficacy obtained upon co-grafting ofLS174T-fluc2 cells with human PBMCs at an effector:target ratio (E:T)5:1 are displayed in FIGS. 3A and 3B.

When administered one day after tumor/PBMCs co-grafting, both doses ofCEA TCB (0.5 and 2.5 mg/kg) induced tumor regression (FIG. 3A). Whenadministered seven days after the co-grafting, only the higher dose (2.5mg/kg) induced tumor regression, whereas the lower dose (0.5 mg/kg) wasunable to induce tumor regression but led to tumor growth control andtumor stasis (FIG. 3B).

The histological analysis of tumors collected at termination of the sameexperiment confirmed that both early (Day 1) treatment with 0.5 mg/kgCEA TCB and delayed (Day 7) treatment with 2.5 mg/kg CEA TCB led tocomplete elimination of CEA-expressing tumor cells and resulted inprominent T-cell infiltration into tumors (not shown). Whereas high-CEAexpression on tumor cells along with variable T-cell infiltration wasdetected in control tumors (vehicle and untargeted MCSP TCB), CEA TCBtreatment (0.5 and 2.5 mg/kg) led to a complete elimination ofCEA-expressing tumor cells along with formation of necrotic/fibroticareas filled with infiltrating T-cells (not shown). The staining of thesame tumors with anti-PD-L1 antibody demonstrated a strong induction ofintra-tumoral PD-L1 expression upon CEA TCB-treatment as compared tovehicle control (FIGS. 4A-4F).

Xenograft Experiment with IP Transfer of Effector Cells

The in vivo efficacy of CEA TCB was also evaluated in xenograft models,in which tumor cells were injected SC followed by IP transfer of PBMCsonce tumors reached a palpable mass. CEA TCB (2.5 mg/kg IV bi-weekly)led to significant tumor regression (reduction of tumor weight, FIG. 5)and to an increase in T-cell infiltration into tumors (flow cytometryanalysis (FIG. 6) and histology (not shown)), with T-cells havingactivated phenotype (as shown by flow cytometry analysis indicatingupregulation of CD25 and CD69 activation markers, not shown). This wasin contrast to the vehicle (PBS) and MCSP TCB (untargeted control),where only a low number of T-cells infiltrated tumors and showed aresting phenotype.

Additional experiments studying PD-1 and CD69 biomarkers showed that,compared to vehicle, CEA TCB induced up-regulation of the earlyactivation marker CD69 and of PD-1 (FIG. 7L, arrow) on tumorinfiltrating T-cells. In contrast, peripheral blood T-cells showed aresting phenotype in both groups (FIGS. 7A-7F).

The staining of the same tumors with anti-PD-L1 antibody showed a stronginduction of intra-tumor PD-L1 expression upon CEA TCB-treatment ascompared to vehicle control.

Xenograft Experiment in Fully Humanized Mice

The antitumor activity of CEA TCB was assessed in vivo in a humanizedmouse model bearing the CEA-expressing MKN45 cancer cell linesubcutaneously.

CEA TCB was injected IV (2.5 mg/kg, twice/week) on Day 11 after tumorcell injection, and tumor growth was measured twice weekly by caliper.FIG. 8 shows individual tumor volumes at study termination; astatistically significant (p<0.05) difference in tumor volume was found.

In addition to tumor volume measurements, tumors, blood, spleen, andlymph nodes were collected at study termination (corresponding to 32days after tumor cell injection) and analyzed by flow cytometry. CEA TCBtreatment resulted in a significant increase of human T-cell frequenciesin the tumor and changed the ratio of CD8+/CD4+ T-cells in favor of CD8+T-cells (FIGS. 9A and 9B). At the same time, both CD4+ and CD8+ T-cellsexhibited an activated phenotype in the CEA TCB-treated tumors shown byup-regulation of CD69 and PD-1 expression on intratumoral CD4+ and CD8+T-cells (FIGS. 9C and 9D). CD8+ T-cells revealed an up-regulation of theintracellular Granzyme B (GZB) expression as well as a higherproliferation rate (Ki-67 marker). Analyses of spleen and blood samplesof CEA TCB-treated animals did not show any difference in terms ofT-cell frequencies, T-cell ratio, or activation status as compared tovehicle.

Thus, the immunological changes triggered by CEA TCB treatment weretumor-specific indicating that the cross-linking and activation of humanT-cells occurs exclusively in CEA expressing tumors and not in otherareas that are negative for CEA like blood and spleen.

Histological analysis of tumors collected at study termination confirmedthe results of flow cytometry (not shown). Whereas human CEA wasexpressed in tumors of both vehicle and CEA TCB-treated groups, the sizeof tumor was significantly reduced by CEA TCB treatment. Moreover,vehicle tumors showed low infiltration of human immune cells that weremostly confined to stromal areas and the tumor border, whereas the CEATCB-treated groups displayed a strong immune cell infiltration throughthe whole tumor area and not only in the periphery. In addition, humanT-cells displayed increased expression of GZB within tumors. Thestaining of the same tumors with anti-PD-L1 antibody demonstrated astrong induction of intra-tumoral PD-L1 expression upon CEA TCBtreatment compared with the vehicle control (FIGS. 10A-10F).

Example 3: Assessment of In Vivo Anti-Tumor Activity Upon Combination ofCEA TCB with Anti-PD-L1 Blocking Antibody in Gastric Carcinoma MouseModel

The anti-tumor efficacy of CEA TCB in combination with anti-human PD-L1blocking antibody was assessed in fully humanized mice bearing thegastric carcinoma tumor cell line (MKN45).

An anti-mouse PD-L1 surrogate antibody based on the YW243.55.570 PD-L1antibody described in WO 2010/077634 (sequence shown in FIG. 11) wasgenerated for use in vivo tumor models. This antibody contained a DAPGmutation to abolish FcγR interaction. The variable region ofYW243.55.570 was attached to a murine IgG1 constant domain with DAPG Fcmutations.

The polypeptide sequences of YW243.55.570 PD-L1 muIgG1 are as follows:

  YW243.55.S70 PD-L1 muIgG1 DAPG HC (SEQ ID NO: 36):EVQLVESGGGLVQPGGSLRLSCAASGFTFSDSWIHWVRQAPGKGLEWVAWISPYGGSTYYADSVKGRFTISADTSKNTAYLQMNSLRAEDTAVYYCARRHWPGGFDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSTWPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDAPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFGAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMDTDGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEKSLSHSPGK YW243.55.S70 PD-L1 muIgG1 LC (SEQ ID NO:37): DIQMTQSPSSLSASVGDRVTITCRASQDVSTAVAWYQQKPGKAPKLLIYSASFLYSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYLYHPATFGQGTKVEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC

In summary, 1×10⁶ MKN45 tumor cells were injected sub-cutaneously infully humanized NOG mice. 7 days after tumor cell injection, mice wererandomized in four groups: the first group received phosphate-buffersaline (PBS, vehicle) as control, the second group received CEA TCB (atthe dose of 2.5 mg/kg, administered twice a week for 7 weeks, 15administrations in total), the third group received anti-PD-L1 antibody(at the dose of 10 mg/kg, administered once a week for 7 weeks, 8administrations in total), and the fourth group received a combinationof CEA TCB and anti-PD-L1 antibody, administered concomitantly with therespective dose and schedules used in the single therapeutic groups for7 weeks (8 administrations of anti-PD-L1 antibody 10 mg/kg given once aweek and 15 administrations of CEA TCB 2.5 mg/kg given twice a week).

The results of this study show a significant increase in anti-tumoractivity upon combination of CEA TCB with anti-PD-L1 blocking antibodyas compared to CEA TCB single agent (FIGS. 11A and 11B). Treatment withanti-PD-L1 antibody as single agent did not show any anti-tumoractivity. No overt test item-related toxicities were observed followingtreatment with the combination.

A second study was designed to assess whether, after a first linetreatment with CEA TCB monotherapy, the combination of CEA TCB witha-PD-L1 could exert superior anti-tumor activity than continuingtreatment with CEA TCB as monotherapy.

For this study, 1×10⁶ MKN45 tumor cells were injected sub-cutaneously infully humanized NOG mice. 10 days after tumor cell injection, micereceived either phosphate-buffer saline (PBS, vehicle) as control, orCEA TCB (at the dose of 2.5 mg/kg, administered twice a week for 4weeks, 7 administrations in total). At study day 34, CEA TCB-treatedmice were randomized into two sub-groups: one continued receiving CEATCB administration (2.5 mg/kg, twice a week), the other received CEA TCB(2.5 mg/kg, twice a week) in combination with anti-PD-L1 antibody (10mg/kg, once a week) for 5 weeks (11 administrations in total for CEATCB, and 6 administrations in total for anti-PD-L1 antibody).

The results clearly show that CEA TCB+anti-PD-L1 combination therapy assecond line treatment mediates significant superior anti-tumor activitythan CEA TCB monotherapy (FIG. 12).

Example 4: Assessment of In Vivo Anti-Tumor Activity Upon Combination ofCEA TCB with Anti-PD-L1 Blocking Antibody in Colorectal Carcinoma MouseModel

The anti-tumor efficacy of CEA TCB in combination with anti-PD-L1blocking antibody was also assessed in fully immunocompetent micebearing the colorectal carcinoma tumor cell line MC38 engineeredin-house to express human CEA on the cell surface (MC38-huCEA).

In summary, 0.5×10⁶ MC38-huCEA tumor cells were injected sub-cutaneouslyin fully immunocompetent C57BL/6 transgenic for human CEA (huCEA Tgmice). 17 days after tumor cell injection, mice were randomized in fourgroups for first line treatment: the first group receivedphosphate-buffer saline (PBS, vehicle) as control, the second groupreceived CEA TCB (at the dose of 2.5 mg/kg, administered twice a weekfor 4 weeks, 7 administrations in total), the third group received ananti-mouse PD-L1 antibody (at the dose of 10 mg/kg, administered once aweek for 4 weeks, 4 administrations in total), and the fourth groupreceived a combination of CEA TCB and anti-PD-L1 antibody, administeredconcomitantly with the respective dose and schedules used in the singletherapeutic groups for 4 weeks (4 administrations of anti-PD-L1 10 mg/kggiven once a week and 7 administrations of CEA TCB 2.5 mg/kg given twicea week).

The results show a significant increase in anti-tumor activity uponcombination of CEA TCB with anti-PD-L1 blocking antibody as compared toCEA TCB and anti-PD-L1 single agents (FIGS. 13A and 13B). Moreover, atstudy day 42, 4 out of 9 mice from the combination treated group weretumor free, while no tumor-free mice were observed upon CEA TCBtreatment, and only 1 out of 8 tumor-free mice were observed uponanti-PD-L1 therapy.

No overt test item-related toxicities were observed following treatmentwith the combination.

At study day 42, sub-cutaneous tumors were surgically removed from allgroups and mice were left untreated for 5 weeks. After this restingperiod, first line-treated mice plus two groups of naïve huCEA Tg mice(one left untouched, and the other previously submitted to a surgicalcut on the flank to mimic surgical intervention) were injectedsub-cutaneously with 0.5×10⁶ MC38-huCEA tumor cell lines on the oppositeflank. No therapy injection was applied to any of the groups, whiletumor growth was monitored. The results show that in all first linetreated mice, tumor protection upon tumor rechallenge was observed, withsignificant tumor control as compared to naïve mice. The group who hadreceived the combination of CEA TCB with anti-PD-L1 as first linetreatment, showed 3 out of 8 tumor-free mice at study day 37 upon tumorrechallenge. These data indicate that in the MC38-huCEA model, thecombination of CEA TCB with anti-PD-L1 antibody not only improved firstline treatment response, but also improved anti-tumor protection uponsubsequent tumor challenge.

Example 5: An Open-Label, Multi-Center, Dose Escalation and ExpansionPhase IB Study to Evaluate the Safety, Pharmacokinetics and TherapeuticActivity of CEA TCB in Combination with Atezolizumab in Patients withLocally Advanced and/or Metastatic CEA-Positive Solid Tumors

Data discussed above demonstrate that CEA TCB and atezolizumab actsynergistically in their anti-cancer properties and collectively theircombination could provide meaningful clinical benefit in patients withcancer.

An open-label, multi-center, dose escalation and expansion Phase Ibclinical study of CEA TCB in combination with atezolizumab is conducted.Each treatment cycle is 21 days in duration and consists of IV infusionsof CEA TCB given weekly (QW) (±1 day) in combination with atezolizumabgiven every 3 weeks (Q3W) (±2 days). Patients receive atezolizumab (1200mg fixed dose) IV on Day 1 of each cycle, followed by CEA TCB at leastone hour later, given IV on Day 1, Day 8 and Day 15 of each cycle.

For CEA TCB, the starting dose is 5 mg administered on a QW schedule, tobe administered at least one hour after the administration of 1200 mg ofatezolizumab when they are both administered on the same day (Day 1 ofeach cycle).

Patients are treated until loss of clinical benefit, unacceptabletoxicities, or withdrawal of consent. In case one of the treatments ispermanently discontinued, treatment with the other drug alone may becontinued.

Key eligibility criteria include confirmed locally advanced and/ormetastatic solid tumor in patients who have progressed on a standardtherapy, are intolerant to standard therapy, and/or are non-amenable tostandard therapy; Eastern Cooperative Oncology Group (ECOG) PerformanceStatus (PS) 0-1; and locally confirmed CEA expression in tumor tissue(≥50% of tumor cells staining with at least moderate to high intensityof CEA expression for all solid tumors with exception of NSCLC≥20% oftumor cells staining with at least moderate to high intensity of CEAexpression) or centrally confirmed CEA expression.

Primary objectives of this study are safety and tolerability, withsecondary objectives of PK and clinical activity (objective overallresponse rate (ORR), disease control rate (DCR; defined as response rate[RR]+stable disease rate [SDR]), progression-free survival (PFS) andoverall survival (OS) data according to Response Evaluation Criteria inSolid Tumors (RECIST), Version 1.1 criteria and modified RECISTcriteria).

Tumor response is evaluated according to RECIST v1.1 and modified RECISTcriteria using unidimensional measurement such as computed tomography(CT) scan or magnetic resonance imaging.

Results

As of 15 Nov. 2016, 30 patients have been treated at the following dosecohort levels: 5 mg of CEA TCB QW+1200 atezolizumab Q3W: 4 patients

10 mg of CEA TCB QW+1200 atezolizumab Q3W: 4 patients

20 mg of CEA TCB QW+1200 atezolizumab Q3W: 4 patients

40 mg of CEA TCB QW+1200 atezolizumab Q3W: 6 patients

80 mg of CEA TCB QW+1200 atezolizumab Q3W: 5 patients

160 mg of CEA TCB QW+1200 atezolizumab Q3W: 7 patients

The combination treatment was at current dose levels (up to 160 mg CEATCB QW+1200 mg atezolizumab Q3W) safe and well tolerated. No patientswere discontinued due to a treatment related safety event.

All patients in this study had an FDG PET scan tumor assessment at week4 and CT scan tumor assessment at week 8 and then every 8 weeks.

At 20 mg CEA TCB, 2/4 patients have experienced prolonged stable diseaseaccording to RECIST criteria. One patient with Pancreas adenocarcinomahas been in the study for 32 weeks, the patient experienced stablemetabolic response assessed by FDG PET at week 4. The second patient, agastro-esophageal junction adenocarcinoma with MSI instability has beenin the study for 30 weeks. This patient experienced a partial metabolicresponse at week 4 FDG PET assessment.

At 40 mg CEA TCB, 1/6 patients experienced prolonged stable diseaseaccording to RECIST criteria and it's been on study for 20 weeks. Thispatient, a colorectal carcinoma patient, was previously treated in themonotherapy trial (BP29541) and received 31 cycles of treatment.

At 80 mg CEA TCB, 5/5 patients experienced tumor volume reduction atweek 8 CT scan and metabolic partial response at week 4 FDG PET scan. Inthis cohort 4/5 patients were colorectal carcinoma patients and 1patient with ampulla Vater carcinoma.

In summary, the clinical data generated so far in this trial showedpromising relevant clinical antitumor activity in 2/4 patients treatedat 20 mg CEA TCB+1200 mg atezolizumab, 1/6 patients treated at 40 mg CEATCB+1200 mg atezolizumab, and 5/5 patients treated at 80 mg CEA TCB+1200mg atezolizumab.

The data discussed above regarding the 80 mg CEA TCB cohort in apopulation that do not respond to anti PD-1 or anti PD-L1 treatment,supports that CEA TCB and atezolizumab act synergistically in theiranti-cancer properties and collectively their combination could providemeaningful clinical benefit in patients with cancer.

1-74. (canceled)
 75. A method for treating or delaying progression ofcancer in an individual comprising administering to the individual aneffective amount of an anti-PD-1 antagonist antibody and ananti-CEA/anti-CD3 bispecific antibody, wherein the anti-CEA/anti-CD3bispecific antibody comprises: (i) a first antigen-binding domain whichis a Fab molecule that binds to CD3, wherein the first antigen-bindingdomain comprises a heavy chain variable region (V_(H)CD3) comprising anHVR-H1 comprising the amino acid sequence of SEQ ID NO: 44, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 45, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 46; and a light chainvariable region (V_(L)CD3) comprising an HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 47, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 48, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 49; (ii) a second antigen-binding domain and athird antigen-binding domain, each of which is a Fab molecule that bindsto CEA, wherein the second antigen-binding domain and the thirdantigen-binding domain each comprises a heavy chain variable region(V_(H)CEA) comprising an HVR-H1 comprising the amino acid sequence ofSEQ ID NO: 38, an HVR-H2 comprising the amino acid sequence of SEQ IDNO: 39, and an HVR-H3 comprising the amino acid sequence of SEQ ID NO:40; and a light chain variable region (V_(L)CEA) comprising an HVR-L1comprising the amino acid sequence of SEQ ID NO: 41, an HVR-L2comprising the amino acid sequence of SEQ ID NO: 42, and an HVR-L3comprising the amino acid sequence of SEQ ID NO: 43; wherein the firstantigen-binding domain is a crossover Fab molecule wherein the variabledomains or the constant domains of the Fab heavy and light chain areexchanged, and each of the second antigen-binding domain and the thirdantigen binding domain is a conventional Fab molecule; and (iii) an Fcdomain comprising a first subunit and a second subunit capable of stableassociation, wherein the second antigen-binding domain is fused at theC-terminus of the Fab heavy chain to the N-terminus of the Fab heavychain of the first antigen-binding domain, and the first antigen-bindingdomain is fused at the C-terminus of the Fab heavy chain to theN-terminus of the first subunit of the Fc domain, and wherein the thirdantigen-binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the second subunit of the Fc domain.
 76. The methodof claim 75, wherein the anti-PD-1 antagonist antibody inhibits thebinding of PD-1 to its ligand binding partners.
 77. The method of claim76, wherein the anti-PD-1 antagonist antibody inhibits the binding ofPD-1 to PD-L1.
 78. The method of claim 76, wherein the anti-PD-1antagonist antibody inhibits the binding of PD-1 to PD-L2.
 79. Themethod of claim 76, wherein the anti-PD-1 antagonist antibody inhibitsthe binding of PD-1 to both PD-L1 and PD-L2.
 80. The method of claim 75,wherein the anti-PD-1 antagonist antibody is selected from the groupconsisting of MDX 1106 (nivolumab), MK-3475 (pembrolizumab), MEDI-0680(AMP-514), PDR001, REGN2810, and BGB-108.
 81. The method of claim 75,wherein the V_(H)CD3 comprises the amino acid sequence of SEQ ID NO: 50and/or the V_(L)CD3 comprises the amino acid sequence of SEQ ID NO: 51.82. The method of claim 75, wherein the V_(H)CEA comprises the aminoacid sequence of SEQ ID NO: 34 and/or the V_(L)CEA comprises the aminoacid sequence of SEQ ID NO:
 35. 83. The method of claim 75, wherein theFc domain is an IgG Fc domain.
 84. The method of claim 83, wherein theFc domain is an IgG₁ Fc domain or an IgG₄ Fc domain.
 85. The method ofclaim 75, wherein the Fc domain is a human Fc domain.
 86. The method ofclaim 75, wherein the Fc domain is a human IgG₁ Fc domain.
 87. Themethod of claim 75, wherein the Fc domain comprises a modificationpromoting the association of the first subunit and the second subunit ofthe Fc domain.
 88. The method of claim 87, wherein in the CH3 domain ofthe first subunit of the Fc domain an amino acid residue is replacedwith an amino acid residue having a larger side chain volume, therebygenerating a protuberance within the CH3 domain of the first subunitwhich is positionable in a cavity within the CH3 domain of the secondsubunit, and in the CH3 domain of the second subunit of the Fc domain anamino acid residue is replaced with an amino acid residue having asmaller side chain volume, thereby generating a cavity within the CH3domain of the second subunit within which the protuberance within theCH3 domain of the first subunit is positionable.
 89. The method of claim75, wherein the Fc domain exhibits reduced binding affinity to an Fcreceptor and/or reduced effector function, as compared to a native IgG₁Fc domain.
 90. The method of claim 75, wherein the Fc domain comprisesone or more amino acid substitutions that reduces binding to an Fcreceptor and/or effector function.
 91. The method of claim 90, whereinsaid one or more amino acid substitutions is at one or more positionsselected from the group of L234, L235, and P329 (EU numbering).
 92. Themethod of claim 75, wherein each subunit of the Fc domain comprises theamino acid substitutions L234A, L235A, and P329G (EU numbering).
 93. Themethod of claim 75, wherein the cancer is a CEA-positive cancer.
 94. Themethod of claim 75, wherein the cancer is colon cancer, lung cancer,ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer,endometrial cancer, breast cancer, kidney cancer, esophageal cancer, orprostate cancer.
 95. The method of claim 94, wherein the colon cancer iscolorectal cancer.
 96. The method of claim 75, wherein the individualhas cancer or has been diagnosed with cancer.
 97. The method of claim75, wherein cancer cells in the individual express PD-L1.
 98. The methodof claim 75, wherein the anti-CEA/anti-CD3 bispecific antibody isadministered at a dose of about 5 mg to about 100 mg every week.
 99. Themethod of claim 75, wherein the individual is refractory to achemotherapeutic agent treatment.
 100. The method of claim 75, whereinthe individual is intolerant of a chemotherapeutic agent treatment. 101.The method of claim 75, wherein the treatment results in a response inthe individual.
 102. The method of claim 75, wherein the anti-PD-1antagonist antibody and the anti-CEA/anti-CD3 bispecific antibody areadministered intravenously, intramuscularly, subcutaneously, topically,orally, transdermally, intraperitoneally, intraorbitally, byimplantation, by inhalation, intrathecally, intraventricularly, orintranasally.
 103. The method of claim 75, further comprisingadministering a chemotherapeutic agent to the individual.
 104. A methodof enhancing immune function in an individual having cancer comprisingadministering to the individual an effective amount of an anti-PD-1antagonist antibody and an anti-CEA/anti-CD3 bispecific antibody,wherein the anti-CEA/anti-CD3 bispecific antibody comprises: (i) a firstantigen-binding domain which is a Fab molecule that binds to CD3,wherein the first antigen-binding domain comprises a heavy chainvariable region (V_(H)CD3) comprising an HVR-H1 comprising the aminoacid sequence of SEQ ID NO: 44, an HVR-H2 comprising the amino acidsequence of SEQ ID NO: 45, and an HVR-H3 comprising the amino acidsequence of SEQ ID NO: 46; and a light chain variable region (V_(L)CD3)comprising an HVR-L1 comprising the amino acid sequence of SEQ ID NO:47, an HVR-L2 comprising the amino acid sequence of SEQ ID NO: 48, andan HVR-L3 comprising the amino acid sequence of SEQ ID NO: 49; (ii) asecond antigen-binding domain and a third antigen-binding domain, eachof which is a Fab molecule that binds to CEA, wherein the secondantigen-binding domain and the third antigen-binding domain eachcomprises a heavy chain variable region (V_(H)CEA) comprising an HVR-H1comprising the amino acid sequence of SEQ ID NO: 38, an HVR-H2comprising the amino acid sequence of SEQ ID NO: 39, and an HVR-H3comprising the amino acid sequence of SEQ ID NO: 40; and a light chainvariable region (V_(L)CEA) comprising an HVR-L1 comprising the aminoacid sequence of SEQ ID NO: 41, an HVR-L2 comprising the amino acidsequence of SEQ ID NO: 42, and an HVR-L3 comprising the amino acidsequence of SEQ ID NO: 43; wherein the first antigen-binding domain is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged, and each of thesecond antigen-binding domain and the third antigen binding domain is aconventional Fab molecule; and (iii) an Fc domain comprising a firstsubunit and a second subunit capable of stable association, wherein thesecond antigen-binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen-binding domain, and the first antigen-binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third antigen-binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe second subunit of the Fc domain.
 105. The method of claim 104,wherein CD8 T cells in the individual have enhanced priming, activation,proliferation, and/or cytolytic activity relative to prior to theadministration of the anti-PD-1 antagonist antibody and theanti-CEA/anti-CD3 bispecific antibody.
 106. The method of claim 104,wherein the number of CD8 T cells is elevated relative to prior toadministration of the anti-PD-1 antagonist antibody and theanti-CEA/anti-CD3 bispecific antibody.
 107. The method of claim 106,wherein the CD8 T cells are antigen-specific CD8 T cells.
 108. Themethod of claim 104, wherein Treg function is suppressed relative toprior to the administration of the anti-PD-1 antagonist antibody and theanti-CEA/anti-CD3 bispecific antibody.
 109. The method of claim 104,wherein T cell exhaustion is decreased relative to prior to theadministration of the anti-PD-1 antagonist antibody and theanti-CEA/anti-CD3 bispecific antibody.
 110. A method for treating ordelaying progression of cancer in an individual comprising administeringto the individual an effective amount of an anti-PD-1 antagonistantibody and an anti-CEA/anti-CD3 bispecific antibody, wherein theanti-CEA/anti-CD3 bispecific antibody comprises: (i) a firstantigen-binding domain which is a Fab molecule that binds to CD3,wherein the first antigen-binding domain comprises a heavy chainvariable region (V_(H)CD3) comprising the amino acid sequence of SEQ IDNO: 50 and a light chain variable region (V_(L)CD3) comprising the aminoacid sequence of SEQ ID NO: 51; (ii) a second antigen-binding domain anda third antigen-binding domain, each of which is a Fab molecule thatbinds to CEA, wherein the second antigen-binding domain and the thirdantigen-binding domain each comprises a heavy chain variable region(V_(H)CEA) comprising the amino acid sequence of SEQ ID NO: 34 and alight chain variable region (V_(L)CEA) comprising the amino acidsequence of SEQ ID NO: 35; wherein the first antigen-binding domain is acrossover Fab molecule wherein the variable domains or the constantdomains of the Fab heavy and light chain are exchanged, and each of thesecond antigen-binding domain and the third antigen binding domain is aconventional Fab molecule; and (iii) an Fc domain comprising a firstsubunit and a second subunit capable of stable association, wherein thesecond antigen-binding domain is fused at the C-terminus of the Fabheavy chain to the N-terminus of the Fab heavy chain of the firstantigen-binding domain, and the first antigen-binding domain is fused atthe C-terminus of the Fab heavy chain to the N-terminus of the firstsubunit of the Fc domain, and wherein the third antigen-binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe second subunit of the Fc domain.
 111. The method of claim 110,wherein the cancer is a CEA-positive cancer.
 112. The method of claim111, wherein the CEA-positive cancer is colon cancer, lung cancer,ovarian cancer, gastric cancer, bladder cancer, pancreatic cancer,endometrial cancer, breast cancer, kidney cancer, esophageal cancer, orprostate cancer.
 113. The method of claim 112, wherein the colon canceris colorectal cancer.
 114. A method of enhancing immune function in anindividual having cancer comprising administering to the individual aneffective amount of an anti-PD-1 antagonist antibody and ananti-CEA/anti-CD3 bispecific antibody, wherein the anti-CEA/anti-CD3bispecific antibody comprises: (i) a first antigen-binding domain whichis a Fab molecule that binds to CD3, wherein the first antigen-bindingdomain comprises a heavy chain variable region (V_(H)CD3) comprising theamino acid sequence of SEQ ID NO: 50 and a light chain variable region(V_(L)CD3) comprising the amino acid sequence of SEQ ID NO: 51; (ii) asecond antigen-binding domain and a third antigen-binding domain, eachof which is a Fab molecule that binds to CEA, wherein the secondantigen-binding domain and the third antigen-binding domain eachcomprises a heavy chain variable region (V_(H)CEA) comprising the aminoacid sequence of SEQ ID NO: 34 and a light chain variable region(V_(L)CEA) comprising the amino acid sequence of SEQ ID NO: 35; whereinthe first antigen-binding domain is a crossover Fab molecule wherein thevariable domains or the constant domains of the Fab heavy and lightchain are exchanged, and each of the second antigen-binding domain andthe third antigen binding domain is a conventional Fab molecule; and(iii) an Fc domain comprising a first subunit and a second subunitcapable of stable association, wherein the second antigen-binding domainis fused at the C-terminus of the Fab heavy chain to the N-terminus ofthe Fab heavy chain of the first antigen-binding domain, and the firstantigen-binding domain is fused at the C-terminus of the Fab heavy chainto the N-terminus of the first subunit of the Fc domain, and wherein thethird antigen-binding domain is fused at the C-terminus of the Fab heavychain to the N-terminus of the second subunit of the Fc domain.
 115. Themethod of claim 114, wherein the cancer is a CEA-positive cancer. 116.The method of claim 115, wherein the CEA-positive cancer is coloncancer, lung cancer, ovarian cancer, gastric cancer, bladder cancer,pancreatic cancer, endometrial cancer, breast cancer, kidney cancer,esophageal cancer, or prostate cancer.
 117. The method of claim 116,wherein the colon cancer is colorectal cancer.